Identification and use of novopeptides for the treatment of cancer

ABSTRACT

Compositions, methods, systems, apparatus and/or articles of manufacture are disclosed for reducing the susceptibility of a population and/or members thereof to cancer, which may include anti-cancer vaccines, components thereof which may include novopeptides, and methods relating thereto.

This application is a continuation-in-part of U.S. non-provisionalapplication Ser. No. 13/052,490, filed Mar. 21, 2011, issued as U.S.Pat. No. 8,796,414 on Aug. 5, 2014, which is a division of U.S.non-provisional application Ser. No. 12/280,389, filed Jan. 15, 2009,which is a U.S. national phase application of PCT internationalapplication No. PCT/US2007/062920 filed Feb. 27, 2007, which claims thebenefit of U.S. Provisional Application No. 60/777,534, filed on Feb.27, 2006; this application claims the benefit of each of the foregoingand each is incorporated herein by reference in its entirety. Theinstant application contains a sequence listing which has been submittedin ASCII format via EFS-Web and is hereby incorporated by reference inits entirety. Said ASCII copy, created on Aug. 3, 2012, is named SAJ-002seq_listing_140803.txt and is 237,551 bytes in size.

BACKGROUND

It is estimated that in 2004 more than 2.4 million new cancer cases willbe diagnosed in the U.S. and more than 1 million are expected to be skincancers. Of those individuals with skin cancer, 96,000 will be diagnosedwith melanoma (4% of newly diagnosed cancers), the most deadly form ofskin cancer. Furthermore, the incidence of melanoma continues toincrease faster than any other cancer. The stochastic nature of 90 to95% of all cancers means that everyone is at risk of developing acancer. In the United States, men have a 50% lifetime risk of developingcancer, while women have a 33% chance (ACS, 2004). With an annualmortality rate of ˜563,700 per year, cancer is the second leading causeof death in the United States.

Vaccination against cancer has been proposed for treatment, andoccasionally prevention, of cancer, and considerable research effort hasbeen devoted to the exploration of a variety of cancer vaccinationstrategies. The goal of finding vaccine compositions and treatmentmethods that are capable of reliably and predictably overcomingtolerance and setting in motion an immune response against tumor cellswithout inducing autoimmunity has, until now, proved elusive. It isnevertheless clear that cancerous cells have characteristics that can berecognized by the immune system, as demonstrated by experiments in whichmice vaccinated with various kinds of tumor cell preparations showprotection from tumor challenge. Antigens that are expressed in or bytumor cells are referred to as “tumor associated antigens” (“TAA's”). Aparticular TAA may or may not also be expressed in non-cancerous cells;TAA's that are not expressed or rarely expressed in non-cancerous cells,or whose expression in non-cancerous cells is sufficiently reduced incomparison to that in cancerous cells that an immune response inducedupon vaccination is reasonably specific to cancerous cells, are referredto as “tumor specific antigens” (“TSA's”).

Over the past two decades, many labs have devised numerous techniqueswhich aim to turn the patient's immune system against a pre-existingtumor (Berzofsky et al., 2004). These include the use of whole cells,peptides, genetically modified tumor cells, heat-shock proteins orapoptotic tumor cells to stimulate the host's immune system to respondto antigens that are characteristic of cancer cells. Arguably the mostelegant approach to cancer vaccination is to use vaccine formulationscomposed of known and defined TAA's, since this will maximizespecificity. Functionally, TAA's may be classed as self and non-selfSelf TAA's are derived from non-mutated genes whose expression islimited to selected normal tissues or to overexpressed proteins. Whilemost TAA's identified to date belong to this self class, there are twolarge potential problems associated with such antigens: autoimmunity andtolerance. Non-self TAA's are expressed exclusively or predominantly bycancer cells, and can be thought of as tumor-specific antigens (TSA's).TSA's can originate either exogenously (such as those derived from viralproteins in virally-associated tumors) or endogenously. Mutation-derivedTSA's can arise from point mutations, translocations, and exonmis-splicing. Unlike self TAA's, TSA's pose greatly reduced risk ofautoimmunity and tolerance.

SUMMARY

In general, disclosed herein are embodiments of methods, systems,apparatus, compositions, and/or articles of manufacture for reducing thesusceptibility of a population and/or members thereof to cancer.Disclosed are methods for identifying and immunologically screeningcandidate antigens for inclusion in a prophylactic and/or therapeuticcancer vaccine. Further disclosed is a general class of antigens,referred to herein as novopeptides, as well as several specificembodiments and/or examples thereof, including non-MS novopeptides andFS-novopeptides and others. Disclosed are methods and compositionsrelated to novopeptides for use in diagnosing, preventing and treatingcancer. Also disclosed are embodiments of methods of using novopeptidesto induce an immune response against cancer. Disclosed are vaccineshaving one or more novopeptide components, which may be used, in variousembodiments, for example, prophylactically, or as a therapeutictreatment against existing cancerous cells.

In a first aspect, disclosed are embodiments of synthetic or recombinantpeptide components, which may include novopeptides, for use inanti-cancer vaccines.

In a second aspect, disclosed are embodiments of anti-cancer vaccinesfor prophylactic or therapeutic administration or both, including, forexample, anti-cancer vaccines including peptide components and/ornovopeptides and anti-cancer vaccines including nucleic acids thatencode peptide components and/or novopeptides.

In other aspects, disclosed are embodiments of methods includingadministration of an anti-cancer vaccine to a population and/or memberthereof.

The foregoing summary is intended to provide a brief introduction to thesubject matter of this disclosure and does not in any way limit orcircumscribe the scope of the invention(s) disclosed herein, which scopeis defined by the claims currently appended or as they may be amended,and as interpreted in the light of the entire disclosure, including thedetailed description and drawing figures.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The accompanying drawing figures, which are incorporated in andconstitute a part of this specification, illustrate several embodimentsand together with the description illustrate various embodiments of thedisclosed compositions and methods.

FIGS. 1a and 1b show PCR amplification of novopeptides FS 1-78 (SEQ IDNO: 2) and FS 6-21 (SEQ ID NO: 4), respectively, from various cancerousand non-cancerous cell types.

FIG. 2 demonstrates an embodiment of a method of determining frameshiftfrequency in tumor cells.

FIG. 3 shows an embodiment of a linear expression element construction(LEE).

FIG. 4 shows results of prophylactic immunization of C57B6 mice with FS1-78 on tumor challenge.

FIG. 5 shows results of prophylactic immunization of Balb/c mice with FS1-78 and FS 6-21 on tumor challenge.

FIG. 6 shows the survival curve in response to prophylactic vaccinationby genetic immunization with a pooled vaccine.

FIGS. 7 a, 7 b and 7 c show an example of a comparison of RNA expressionlevels of novopeptides in tumor cells with that in non-cancerous cells.

FIG. 8 shows ELISA results indicating reactivity of serum from 23 cancerpatients to FS 6-21 (SEQ ID NO: 4).

FIG. 9 shows results of an immunological screen via a CTL assay.

FIG. 10 shows immunofluorescence images of anti-FS 6-21 serum applied toB16 and 4T1 tumor cells and pre-immune serum applied to B16 tumor cells.

FIG. 11 shows an animal survival curve in response to therapeuticvaccination with frameshift peptide-encoding sequences.

FIGS. 12a and 12b show tumor progression and survival following tumorchallenge in mice receiving therapeutic vaccination with an embodimentof a novopeptide associated with a frame shift mutation or variation inFS SMC-1A (SEQ ID NO:8).

FIGS. 13a and 13b show tumor progression and tumor count in an exampleembodiment wherein BALB-neuT mice received prophylactic immunizationwith FS RBM peptide (SEQ ID NO:232) or FS THAP2 peptide (SEQ ID NO:238),

FIGS. 14a and 14b show tumor progression and tumor count in an exampleembodiment wherein BALB-neuT mice received prophylactic immunizationwith pooled antigen compositions.

FIG. 15 shows prophylactic inhibition of tumor progression in BALB/C-4T1mice vaccinated with an embodiment of an anti-cancer vaccine.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

The methods and compositions disclosed herein pertain in part to a classof compositions, referred to herein as novopeptides, which are useful ascandidates for cancer vaccines. Herein, “novopeptide” refers to anycomposition, such as, for example, a TSA, comprising a polypeptidehaving at least 8 and no more than 40 amino acids, whose amino acidsequence is encoded by all or part of at least one novopeptide nucleicacid sequence. Thus, for example, a “novopeptide can comprise a TSAhaving 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or40 amino acids or any number of amino acid residues in between. A“novopeptide nucleic acid sequence” means any nucleic acid sequence thatcan be generated from any non-cancerous reference sequence by anovopeptide associated mutation or variation. “Novopeptide” includes anysuch polypeptide regardless of how produced or obtained, whethernaturally occurring, engineered, produced by in vitro translation,synthesized, or produced in any of the many other ways of generatingpolypeptides known to one having ordinary skill in the art. A“novopeptide associated mutation or variation” means one or acombination of any one or more point mutations, frame shift mutations,in-frame insertions or deletions, translocations, improper splicing,post-transcriptional events, variations, or other alterations in anucleic acid sequence from a non-cancerous reference sequence,regardless of whether heritable or not, the effect of which is to causethe amino acid sequence or composition of a polypeptide encoded therebyto differ from that of the non-cancerous reference sequence;“novopeptide associated mutation or variation” expressly includes,without limitation, deviations from non-cancerous reference sequencesoccurring as a result of mis-translation, mis-splicing, or other eventsoccurring at the RNA level. A “non-cancerous reference sequence” meansand includes any nucleic acid sequence occurring in any non-cancerouscell of the organism of interest, whether or not expressed therein. Theterms “cancerous cell” and “cancer cell” mean and include any cellexhibiting cancerous, precancerous, dysplagic or other changescharacteristic of the transformation of a normal cell into a tumor cell,whether or not malignant and whether or not immediately tumorigenic. Itwill be recognized that the ontogeny of cancer typically entails asuccession of cellular events, and that treatment, whether prophylacticor therapeutic, is optimally applied at the earliest possible stage ofthat succession. The methods and compositions disclosed herein areintended to apply not only to conditions that have progressed to thepoint where they are diagnosable as cancer but also to any and allconditions associated with the expression of novopeptides by cells in amanner immunologically distinguishable from normal cells. “Tumor cell”means a cell obtained from or associated with a tumor. “Noncancerouscell” means and includes any cell that is not a cancerous cell or tumorcell. Typically, a novopeptide is a linear polypeptide sequencecomprising naturally occurring amino acids; however, “novopeptide” alsoincludes any other polypeptide composition including a sequence that canbe expressed by a cancerous cell as a result of a novopeptide associatedmutation or variation of a noncancerous reference sequence, whetheroccurring in DNA or RNA, whether or not comprising one or more aminoacids that differ from the naturally occurring amino acids, whether ornot post-translationally modified, and whether or not bonded to orassociated with any one or more other moieties. A FS-novopeptide is anovopeptide whose sequence differs from that of a noncancerous referencesequence in a manner attributable to one or more novopeptide associatedmutations or variations wherein the mutation or variation is a frameshift mutation or variation. A non-MS novopeptide is a novopeptideencoded by a novopeptide nucleic acid sequence that can be generated bya novopeptide associated mutation or variation from a non-cancerousreference sequence that is not a microsatellite sequence.

One interesting technology that has been developed to identifyframeshifts without having to sequence genes is the high-throughputsolid-phase protein truncation test (HTS-PTT) (Gite et al., 2003).However, a problem with this approach is that the user must have one ora few candidate proteins in mind at the outset, which reintroduces theproblem of requiring knowledge about gene function or mechanism. In thepresent method, high-throughput sequencing capabilities andbioinformatics may be used to identify FS cancer vaccine candidates. Incontrast to prior methodology, various embodiments of the methodsdisclosed herein 1) do not require knowledge about gene function orimmunological mechanism, 2) are systematic and amenable tohigh-throughput, and 3) are generalizable to all types of cancer. Noother approach has all three of these characteristics. Furthermore,testing in the melanoma mouse model confirms that these novopeptides areeffective therapeutic vaccines and prophylactic vaccines.

The least explored and potentially most useful subclass of TSA isarguably that caused by frameshifts (FS). One of the consequences oftransformation from a normal cell to a cancer cell is that DNAreplication and RNA processing become more error prone, while DNA repairbecomes less robust. This causes an increase in the frequency of FSmutations or variants where 1 or 2 (or other non-multiple of three) newbases are inserted into or deleted from a gene. When such mutationsoccur in the coding regions of proteins, the resulting shift in readingframes gives rise to the synthesis of truncated genes that have losttheir function. On average at least 20% of the FS variants would encodea new peptide of 9 or more amino acids. Since ˜9 amino acids arerequired to bind in the MHC I pocket for presentation to T cells (e.g.,8, 9, 10, or 11 residues), many of the FS variants could be presented.It will be seen that even short FS variants will present new 9-residuepeptides by virtue of the fusion of wild-type and FS sequences.Furthermore, as these nonsense proteins tend to be very immunogenic andare expressed predominantly (if not exclusively) in tumor cells,FS-derived antigens are ideal vaccine candidates. In addition toframeshifts, an insertion or deletion of a nucleic acid sequence that isa multiple of three will produce an in-frame deletion or insertion.These will also lead to the production of novopeptides since thejunction points will be new peptide sequence.

Relative to oncogenesis, there are at least two classes of mutatedproteins to consider, whether produced by frameshifts or othermechanisms: the first class, oncogenic-related variants, are those thatresult in or contribute to tumor formation or progression. The secondclass, bystander variants, are those that are not involved inoncogenesis but that happen to be altered simply because the cellularmachinery is operating inefficiently or for other reasons. From thepoint of view of developing a vaccine, both are viable as vaccinecandidates.

Disclosed herein are embodiments of methods of screening for atumor-specific antigen, comprising obtaining a tumor cell, extractingRNA from the cell, and assaying for frameshifts. It is understood andherein contemplated that the tumor-specific antigen can be a peptide orprotein.

Disclosed are embodiments of methods of identifying components for aprophylactic cancer vaccine, comprising: identifying novopeptides byinformatics, genomics, proteomics or immunological screens; anddetecting an immune response to the novopeptide that differentiatesbetween tumor and normal cells. The novopeptide so identified can beused to induce a primary immune response.

Disclosed herein are embodiments of methods of identifying a novopeptidethat produces an anti-cancer immune response, comprising identifying anovopeptide by informatics, genomics, proteomics, or immunologicalscreens; and determining that the novopeptide induces an immune responsethat differentiates between tumor cells and normal cells. It isunderstood that the novopeptide of the method can be identified by anyof the methods disclosed herein. Thus, for example, disclosed herein areembodiments of methods, wherein the novopeptide is identified usingcancer genome and expression databases to detect novopeptidespreferentially expressed in tumor cells versus normal cells.Alternatively, disclosed are methods, wherein the novopeptide isidentified using nucleic acid sequencing methods to detect alterationsin DNA and or RNA that lead to the novopeptide. Also disclosed aremethods wherein the novopeptide is identified using mass spectrometry todetect novopeptides that are on the tumor cell surface.

It is understood and contemplated herein that any immunoassay that canmeasure a T cell response can be used in the disclosed methods. Thus,for example, disclosed herein are embodiments of methods of identifyinga novopeptide that produces an anti-cancer immune response comprisingdetermining that the novopeptide induces an immune response, wherein thenovopeptide is identified using immune assays of human cancer patientserum or animal tumor model serum to detect reactivity to thenovopeptide. Also disclosed are methods, wherein the novopeptide isidentified using immune assays of human cancer patient peripheral bloodmononuclear cells (PBMCs) or animal tumor model (PBMCs) to detectreactivity to the novopeptide. As noted above, the immune assay can beany assay known in the art that measures T cell activity. Thus, forexample, the immune assay can be a cytolytic assay such as a 51Crrelease assay, or the assay can measure cytokine production in responseto the peptide such as ELISPOT, ELISA, and Intracellular CytokineStaining. Thus, disclosed herein are embodiments of methods wherein theimmune assay is selected from the group consisting of ELISPOT, ELISA,and Intracellular Cytokine Staining. Antibodies may also be used toidentify T cell activity by binding to T cells specific for anovopeptide. For example, MHC class I and II tetramers, dimers, andtrimers can be used to mark novopeptide specific T cells.

Also disclosed are methods of identifying a novopeptide that induces aprotective immune response to cancer, comprising identifying anovopeptide by informatics (odds ratios of tumor to normals); sequencingcandidate DNA or RNA; performing mass spectrometry on peptides elutedfrom MHCI of tumor cells and normal cells, and detecting the peptidesthat are expressed by tumor cells; and determining whether T-cellsreactive to the novopeptide peptide react with MHCI matched tumor cellsbut not normal cells. It is understood that additional steps may beneeded to identify novopeptides. Thus, disclosed herein are embodimentsof methods, further comprising comparing the peptides eluted from tumorMHCI to a database of all possible novopeptides from the human proteome.It is also understood that antibody responses to a novopeptide can alsobe desirable in therapeutic methods. Therefore, disclosed herein areembodiments of methods of identifying a novopeptide that induces aprotective immune response to cancer, further comprising determining ifantibodies raised to the novopeptide react with tumor cells expressingthe novopeptide and not with normal cells. Also disclosed are methods ofidentifying a novopeptide that induces a protective immune response tocancer, comprising identifying a novopeptide by informatics (odds ratiosof tumor to normals); sequencing candidate DNA or RNA; performing massspectrometry on peptides eluted from MHCI of tumor cells and normalcells, and detecting the peptides that are expressed by tumor cells; anddetermining if antibodies raised to the novopeptide react with tumorcells expressing the novopeptide and not with normal cells.

It is understood and herein contemplated that the disclosed methods ofidentifying novopeptides that produce an anti-cancer immune responsewill produce peptides useful in producing an immune response to cancer.Thus, the novopeptides identified by the methods disclosed herein andthose specifically elucidated can be used as a therapeutic orprophylactic agent to treat or prevent a cancer either alone or incombination with other peptides or known anti-cancer agents. Thus, forexample, the disclosed methods can identify novopeptides that can beused to develop an anti-cancer vaccine. Therefore, disclosed herein arecancer vaccines comprising a novopeptide or nucleic acid encoding anovopeptide that has been identified by any of the methods ofidentifying novopeptides disclosed herein. It is understood and hereincontemplated that such a vaccine can be delivered by any method known inthe art including but not limited to gene gun, as gene vaccine, viralvector or as peptide or peptide fusion to another carrier such as aprotein, sugar, or oil:water emulsion.

The disclosed prophylactic and therapeutic vaccines are suitable foradministration to human and non-human subjects. Thus, disclosed hereinare prophylactic vaccines that are administered to a non-human animalselected from the group consisting of dog, cat, guinea pig, mouse, rat,rabbit, pig, horse, cow, monkey, chimpanzee, or other non-human primateto prevent cancer, or to any other animal susceptible to cancer.

Thus, disclosed herein are embodiments of methods of identifying anovopeptide that induces a protective immune response to cancer,comprising identifying a novopeptide by informatics (odds ratios oftumor to normals); sequencing candidate DNA or RNA; performing massspectrometry on peptides eluted from MHCI of tumor cells and normalcells, and detecting the peptides that are expressed by tumor cells; anddetermining whether T-cells reactive to the novopeptide react with MHCImatched tumor cells but not normal cells. It is understood thatadditional steps may be needed to identify novopeptides. Thus, disclosedherein are embodiments of methods, further comprising comparing thepeptides eluted from tumor MHCI to a database of all possiblenovopeptides from the human proteome. It is also understood thatantibody responses to a novopeptide can also be desirable in therapeuticmethods. Therefore, disclosed herein are embodiments of methods ofidentifying a novopeptide that induces a protective immune response tocancer, further comprising determining if antibodies raised to thenovopeptide react with tumor cells expressing the novopeptide and notwith normal cells. Also disclosed are methods of identifying anovopeptide that induces a protective immune response to cancer,comprising identifying a novopeptide by informatics (odds ratios oftumor to normals); sequencing candidate DNA or RNA; performing massspectrometry on peptides eluted from MHCI of tumor cells and normalcells, and detecting the peptides that are expressed by tumor cells; anddetermining if antibodies raised to the novopeptide react with tumorcells expressing the novopeptide and not with normal cells.

The disclosed methods can also be used in conjunction with animalmodels. Thus, disclosed herein are embodiments of methods of identifyinga novopeptide that produces an anti-cancer immune response, comprisingidentifying a novopeptide by informatics, genomics, proteomics, orimmunological screens; and determining that the novopeptide induces animmune response that differentiates between tumor cells and normalcells, wherein the anticancer immune response of the novopeptide isfurther determined by administering a non-human animal homolog of ahuman novopeptide to the non-human animal in a prophylactic ortherapeutic cancer model; and measuring the anti-cancer effect of thenovopeptide in the animal model of cancer.

It is understood and herein contemplated that any of the disclosedmethods benefit by the distinction and identification of immuneresponses limited to tumor cells (i.e., not present or present at onlylow levels in normal cells). Thus, disclosed herein are embodiments ofmethods, for further identifying the induction of an immune responsethat differentiates between tumor cells and normal cells, wherein humancells are exposed to the novopeptide, and the reactivity of the exposedcells to human cancer cells and normal cells is determined, wherein astronger reactivity against human cancer cells compared to normal cellsindicates a cancer-specific immune response.

Tumor specific antigens can come from many sources. One advantage of thepresent disclosure over previous methods is the identification oftumor-specific antigens in genes previously not associated withoncogenesis (i.e., cancer). For example, one source of tumor-specificantigens is frameshifts of genes. The genes can be oncogenic ornon-oncogenic. A frameshift originating from an oncogene is an“oncogenic-related frameshift,” whereas, a frameshift derived from anon-oncogenic tumor gene is a “bystander frameshift.” Thus, for example,specifically contemplated herein are tumor-specific antigens wherein theantigen is the result of a bystander frameshift in the gene source.

In embodiments, a method for identifying novopeptide vaccine antigenscomprises two major tasks. The first, hereinafter referred to as anovopeptide identification screen, entails identifying novopeptidesand/or novopeptide nucleic acid sequences that are likely to beexpressed and/or are experimentally determined to be expressed in one ormore cancerous cell types. The second task, hereinafter referred to as anovopeptide immunological screen, entails immunological screening of thenovopeptides so identified, or novopeptides encoded by the novopeptidenucleic acid sequences so identified, to evaluate each candidatenovopeptide for suitability as a component of a vaccine.

An important and novel insight underlying the disclosure hereof, andverified by the experiments described below, is that the widely heldassumption that antigens expressed in cancerous cells and minimallyexpressed or not expressed in noncancerous cells are derived fromoncogenes, particularly or exclusively those containing microsatellitesequences, does not withstand scrutiny, in fact, cancer cells canexpress many genes that have undergone a novopeptide associated mutationor variation, resulting in the expression of one or more non-MSnovopeptides or other non-oncogene associated novopeptides by the cell.

The novopeptide identification screen in one aspect relates toidentification of novopeptides that are expressed, or are predicted tobe expressed, in cancerous cells. This can be accomplished in a numberof ways, for example, the methods described by the examples disclosedherein. The method extends to the approaches described herein, which areoffered as examples only and not intended to limit the scope of thedisclosure, as well as any of the other methods known to persons havingordinary skill in the art for identifying peptides, peptide sequences,and/or nucleic acid sequences encoding peptides, that are experimentallydetermined to be expressed or predicted to be expressed in apredetermined cell type and/or that exhibit predeterminedcharacteristics.

One method for performing the novopeptide identification screencomprises generating a library of candidate novopeptide sequences,and/or novopeptide nucleic acid sequences, bioinformatically from aknown genome sequence or subsequence, or from cDNA, mRNA, EST, proteinor peptide sequence, nucleic acid or peptide microarray data, or anyother data from which the sequence encoding any non-cancerous referencesequence can be determined or inferred. At least one non-cancerousreference sequence is extracted from such data. Without limiting thegenerality of the foregoing, and by way of example only, one way ofextracting a non-cancerous reference sequence from such data is toextract the DNA or RNA sequence corresponding to a known gene or openreading frame from available sequence data. Because many novopeptideassociated mutations or variations are the result of events occurring atthe level of RNA processing and/or translation, RNA sequences areanother important source of sequence data for identification ofcandidate novopeptides. Ideally, a non-cancerous reference sequence soextracted is a sequence that, when mutated and fragmented and/orrecombined to form novopeptide nucleic acid sequences, is likely to beexpressed in a cancerous cell; however, novopeptide identification is inpart a trial and error process, so not all non-cancerous referencesequences so extracted will be ideal. Nevertheless, the selection ofnon-cancerous reference sequences can, in appropriate circumstances, beoptimized by any of the methods known to a person having ordinary skillin the art for estimating the likelihood of expression of a sequence,such as, by way of example only, taking into account the locus of thesequence in question with respect to a known promoter and/or otherregulatory elements, and/or taking into account the relationship of thesequence in question to a gene known to be expressed in cancerous cellsof a type for which a vaccine is desired. From each non-cancerousreference sequence extracted from the sequence data, one or morenovopeptide nucleic acid sequences is generated by applying anovopeptide associated mutation or variation and extracting one or moresubsequences affected by the novopeptide associated mutation orvariation and having lengths corresponding to novopeptides of thedesired length. Many other methods for identifying candidate novopeptidesequences from genomic, proteomic, or other similar data will beapparent to a person having ordinary skill in the art. Once a library ofcandidate novopeptide sequences has been generated, physicalnovopeptides can readily be generated therefrom by any of the manymethods known to a person having ordinary skill in the art forsynthesizing or producing physical polypeptides from specifiedsequences, including without limitation and by way of example only, FMOCsynthesis, in vitro translation, and genetically engineered bacterial,phage, or yeast expression systems.

There exist large public databases that contain the sequences of DNA orcDNA from various tumor samples and from normal tissues. The NCI ESTdatabase currently contains more than 41 million entries. The CancerGenome Atlas Project is another source of tumor cell sequence data.Comparison of sequences in the tumor databases to non-cancerousreference sequence open reading frames reveals putative insertions,deletions, mis-splicings, and other variations that can lead totranslation and expression of novopeptides.

The disclosure hereof in one aspect relates to the task of identifyingnovopeptides likely to be expressed in cancer cells and not at all or ata low level in non-cancerous cells, which may, in embodiments, beaccomplished by comparing EST sequences from a tumor database with ESTsequences from a non-tumor related EST database or other sequencedatabase to identify sequences arising from frame shift mutations orvariations. EST sequences are particularly useful because they representsequences known to be expressed, and capture variation occurring at theRNA level that may not be apparent in the corresponding DNA sequence. Insome embodiments, all possible frame shifted sequences are generatedfrom the non-tumor EST database, and the tumor EST database is thensearched for sequences matching the frame shifted sequences sogenerated. The matching sequences found in the tumor EST database arethen ranked for selection taking into account the number of times eachframe shifted sequence appears in the tumor EST database as compared tothe number of times the unshifted noncancerous reference sequenceappears in the non-tumor EST database. Both databases are highlyredundant, being repositories for data from many experiments by manyresearchers, and represent a reasonable sample of expression in tumorand non-tumor cells, respectively. Another factor to be taken intoaccount is the size of the insertion or deletion resulting in aframeshift found in the tumor EST database. Insertions or deletions ofthree or fewer nucleotides have a significant likelihood of being due tosequencing errors, while longer insertions or deletions, particularlythose appearing in multiple EST's deposited from multiple sources, arehighly likely to represent true novopeptides that are actually expressedin tumor cells. It will be noted that the bioinformatic approach justdescribed also provides information useful for selecting novopeptidesthat are expressed in multiple tumor types.

In embodiments, another method for identifying novopeptides expressed intumor cells may entail extracting RNA from tumor cells and sequencingthe RNA so extracted, using any of the methods familiar to a personhaving ordinary skill in the art for extracting and purifying RNA fromcells and determining the sequence of the RNA. An interesting findingupon sequencing genes in human tumor cell lines for frame shift variantsthat are predicted to occur based on the bioinformatic predictionmethods described herein is that many frame shifted sequences terminateat a shorter length than statistically expected. Since three of the 64possible codons are stop codons, one would expect a stop codon to occuron average approximately once every 21 codons, or about once every 63amino acids, if codons were randomly distributed, but many immediateterminations were observed and frame shift variants longer than about 20amino acids were rarely encountered.

Also disclosed herein are embodiments of methods of performing anovopeptide identification screen comprising extracting novopeptides inphysical form from a sample containing known or suspected cancerouscells, and identifying the novopeptides so extracted. A variety ofmethods exist that are capable of extracting any novopeptides that canbe present in or on one or more cells (typically but not necessarilytogether with other substances that can be present in the sampleincluding other cellular proteins and peptides). Several such methodsare known to those of skill in the art, and include without limitationand by way of example only, washing with selected solvents or buffers,acid elution, sonication, and elution from MHC by competition with otherchemical entities having an affinity for MHC. A method may be chosenthat can extract antigens present on the surface of cells in the sample.Methods that preferentially extract antigens displayed in MHC are ofparticular utility since novopeptides expressed by cancerous cells arelikely to be so displayed. Once an extraction containing novopeptideshas been obtained, the novopeptides contained therein can becharacterized and their sequence determined by any of the methods knownto a person having ordinary skill in the art for extracting andsequencing peptides from an inhomogeneous sample. Commonly used methodsinclude without limitation sample separation by chromatographic and/orelectrophoretic means, followed by characterization of the fractionsthus separated, which can be by sequencing methods such as Edmandegradation, or by mass spectroscopy. Other methods exist foridentification of specific sequences using antibody or other probes,including without limitation ELISA and microarray analysis. Aparticularly useful and heretofore unfeasible approach enabled byvarious embodiments of the methods disclosed herein is separation andidentification of novopeptides by liquid chromatography and massspectroscopy (LC-MS/MS). For a novopeptide to be most effective as atarget for a vaccine, it should be presented on the outside of the tumorcells. For T-cell killing of the tumor the peptides should be presentedin the context of an MHC molecule. For anti-tumor antibody binding thenovopeptides need to be accessible on the surface in some form. Massspectrometry allows the direct detection of particular sequences ofpeptides. Identification of novopeptides by MS has heretofore not beenpossible, in part because mass spectrometers having resolutionsufficient to resolve peaks corresponding to novopeptides have onlyrecently become available, and, more importantly, because identificationof novopeptides by MS requires a database of novopeptide sequences andcorresponding masses, and no such database has existed until created atthe inventors' direction for purposes of the methods disclosed herein.In embodiments, such a database can be constructed by assembling a setof candidate novopeptide nucleic acid sequences by any of the methodsfor doing so disclosed herein or known to a person of ordinary skill inthe art, and analyzing each sequence using software (such as, by way ofexample only, BIMAS and/or SYFPEITHI) for predicting the ability of asequence to bind to or be displayed in the MHC types present in thetumor cells from which the novopeptides are being eluted and to identifypreferred 9-mer sequences or subsequences that are capable of beingdisplayed in those MHC types. Spectra corresponding to each preferred9-mer sequence so determined may be generated and compared with spectrameasured via LC-MS/MS using software (such as, by way of example only,Spectrum Mill) suitable for generating spectra from peptide sequences,comparing the spectra so generated with measured spectra, and from suchcomparison assessing whether a peptide sequence corresponds to any ofthe measured spectra. Because novopeptides may be present at low levelsand only one sequence presented, until recently the sensitivity of massspectrometry was not high enough to detect them. It should be noted thatthe method just described can be used both for identification ofcandidate novopeptides and as a screen to support or verify theidentification by one of the other methods described.

Particularly with regard to human cancers, it is useful to performbioinformatic screening of candidate novopeptides for likely HLAcompatibility, since humans are outbred, while laboratory mice are not.It is understood and herein contemplated that the effectiveness of aparticular novopeptide as a vaccine for human use depends in part uponthe ability of the novopeptide to be displayed by the HLA types presentin the human patient to whom it is administered. Vaccine candidatenovopeptides can be assessed for likely ability to be displayed by givenHLA types using algorithms known to those having ordinary skill in theart, such as, for example, those described herein. For vaccination of aparticular human patient, the vaccine should preferably include one ormore novopeptides predicted to have a high probability of binding to atleast one of the HLA types expressed in the cells of the patient. For avaccine intended for non-personalized use in humans, the vaccine shouldinclude one or more novopeptides in each of a number and selection ofHLA types sufficient that a high percentage of individuals in the targetpopulation will have at least one of the HLA types represented in thevaccine, keeping in mind that it is not uncommon for one peptide to bepresented by two or more MHC molecules, thereby reducing the number ofdistinct novopeptides required for a desired level of populationcoverage. It is also useful to take into account the particular tumortypes in which particular novopeptides are expressed or are predicted tobe expressed, the frequency with which those tumor types appear in thetarget population, the urgency of finding effective treatment orprophylaxis for those tumor types (keeping in mind that no effectivetreatments exist for some cancers and that cancer types differ in termsof life expectancy after diagnosis and severity of effects), and anyother criteria deemed important in designing a vaccine. The methods andcompositions disclosed herein enable preferential selection for furthertesting of novopeptides that are expressed in multiple tumors, that aremore commonly occurring, that are more urgently in need of an effectivevaccine, or that meet other criteria.

Also disclosed are embodiments of methods of performing a novopeptideidentification screen comprising comparing the RNA expression level of aparticular novopeptide in tumor cells of the type being targeted to theRNA expression level of the same novopeptide in one or morenon-cancerous cell types. This can be accomplished by any of the methodsknown to a person having ordinary skill in the art for assaying for RNAexpression levels, such as, without limitation and by way of exampleonly, microarray expression analysis, reverse transcriptase PCR, andSAGE analysis. For inclusion in vaccines, novopeptides that are highlyexpressed in tumor cells and minimally expressed or not expressed innon-cancerous cells are preferred. For an effective vaccine, thenovopeptide or a functionally similar peptide should preferably beexpressed in the tumor being targeted, and ideally not in non-cancerouscells, and since some novopeptides are highly differentially expressedin tumor vs. non-cancerous cells and others are not, the RNA expressionlevel screen is useful for optimizing the selection of novopeptides forinclusion in vaccine formulations.

Disclosed herein are embodiments of methods for performing novopeptideimmunological screens. For example, disclosed herein are embodiments ofmethods for immunologically screening for the existence of a B cellresponse to a particular novopeptide comprising assaying for thepresence of antibodies reactive to that novopeptide in serum samplesfrom individuals having a type of cancerous cells predicted to expressthe novopeptide, and for the absence, or presence below a prespecifiedtiter, of such reactive antibodies in serum obtained from one or moreindividuals not having such cancer. The presence in sera of antibodiesreactive to a given novopeptide may be detected and quantified by anELISA assay in which the novopeptide is adsorbed onto a solid surface,serum is applied, and antibodies remaining bound to the novopeptidesafter washing are detected. The initial immune response to variantantigens displayed on naturally occurring tumors is suppression andtolerization due to the absence of the co-regulatory signals requiredfor mounting of an immune response; this has been demonstrated in animalmodels and is probably the case in humans. However, in at least someindividuals, a strong immune response develops late in the tumorprogression process. Therefore, serum antibody reactivity to a candidatenovopeptide, even if detected in the serum of only one or a fewindividuals having the cancer type in question, is strong evidence thatthe novopeptide is expressed in that cancer type.

Disclosed herein are embodiments of methods for immunologicallyscreening for a T cell response to a particular novopeptide comprisingfirst preparing cytotoxic T lymphocytes (“CTL's”) having T cellreceptors specific for the novopeptide as displayed in MHC or HLA. TheseCTL's may then be tested for reactivity against each of (1) cancerouscells, and (2) non-cancerous cells, each having an MHC or HLA typematching that of the MHC or HLA for which the CTL's are specific.

In embodiments, another method for screening of novopeptides entailstesting in a suitable animal model by immunizing with a novopeptide tobe evaluated and observing whether the immunization is effective inproducing a prophylactically or therapeutically beneficial immuneresponse upon challenge with tumor cells, or in an animal having orprone to having a tumor. The response can be assessed by, for example,measuring tumor volume over time, or assessing survival rates, incomparison to non-immunized controls. Example 2 is illustrative of thesemethods.

Any one or more of the screening methods described in the precedingdiscussion can be used to identify novopeptides that are prevalent intumors relative to noncancerous cells. By screening a panel of tumor andnon-cancerous cells it is possible to establish the frequency of anovopeptide in specific tumor types as well as in tumors generally.Further, by screening a novopeptide against the known frequencies of HLAtypes it is possible to establish the percentage of a population thatare likely to respond to the antigen.

As already noted, a second task to which the disclosure hereof isdirected is that of performing a novopeptide immunological screen of thecandidate novopeptides identified via the novopeptide identificationscreen or otherwise. A goal of the novopeptide immunological screen isto determine the suitability of a given candidate novopeptide forinclusion in a therapeutic or prophylactic vaccine. In embodiments, anovopeptide immunological screen can be carried out by employing themethods disclosed in the preceding paragraphs, or by any of the methodsknown to a person having ordinary skill in the art for determining orestimating the likely efficacy and safety of a biomolecule as a vaccinecomponent, singly or in combination and in any appropriate order. In anembodiment, a novopeptide immunological screen entails determiningwhether T-cells made reactive to the novopeptide exclusively ordisproportionately react with cancerous cells but not normal cells. Withregard to B-cell response, an embodiment of a novopeptide immunologicalscreen may entail determining whether antibodies against the novopeptidespecifically react against tumor cells and not normal cells.Novopeptides are inherently relatively unlikely to be expressed innon-cancerous cells, since novopeptides are derived from altered nucleicacid sequences. It is preferable that novopeptide vaccine antigens notbe expressed in non-cancerous cells, since such expression would imply alikelihood of existing tolerance, and since it is preferable that avaccine not produce an immune response against non-cancerous cells.However, the preference for non-expression in non-cancerous cells is nota rigid one, since even treatments that produce undesired side effectscan be therapeutically useful.

Disclosed herein are embodiments of methods and compositions useful inthe formulation of prophylactic and/or therapeutic vaccines to beadministered for the purpose of raising an immune response against tumorcells. The disclosure hereof extends to the composition ofnovopeptide-based vaccines and to methods of administration thereof. Anovopeptide-based vaccine can be prepared and administered in any of theways familiar to persons having ordinary skill in the art, including thevery simple approach of preparing a vaccine comprising a novopeptidedissolved or suspended in a suitable carrier, and administering it onceor at predetermined intervals to the animal or human patient to bevaccinated. However, in embodiments, better success may be had by othermethods, and a particular approach entails genetic immunization usinggene gun technology, in which the vaccine is administered in the form ofa linear expression element encoding the desired novopeptide, asillustrated in the examples below. The composition of a vaccine caninclude both novopeptide and other components. The inclusion of multipledistinct novopeptides can improve the level of imrnunoprotectionconferred, and may be employed to confer imrnunoprotection againstadditional tumor types. Single novopeptides may be found that conferimrnunoprotection against more than one tumor type, but the repertoireof target tumor types can be expanded by inclusion of additionalnovopeptides. The inclusion of multiple novopeptides is of particularutility in vaccines intended for administration in humans, due to theneed for including a number and selection of novopeptides sufficient toensure that at least one novopeptide in the vaccine will be capable ofbeing displayed by at least one HLA type present in each individual in apredetermined percentage of the target population. In embodiments, twoor more novopeptides can be fused into a single entity. In embodiments,novopeptide-based vaccines can include other components familiar to aperson having ordinary skill in the art for improving theimrnunoprotection conferred or otherwise improving the efficacy and/orsafety of the vaccine formulation, including without limitation and byway of example only, adjuvants and hapten carriers.

Experiments have been performed to assess directly the feasibility ofcreating general prophylactic cancer vaccines and therapeutic cancervaccines. In contrast to existing dogma, results from these experimentsindicate that it is possible to immunize prophylactically withnovopeptide vaccines that cross-protect across different tumor types andin different MHC backgrounds. These results show that cancer vaccines donot have to be personalized; can contain a defined set of tumor specificantigens (novopeptides) that cover the majority of human MHCs; and canbe administered prophylactically and so avoid the necessity of delayingtreatment until an individual develops a tumor (at which point thebattle is harder to win) so that a sufficient personalized sample can beobtained to allow formulation of a drug or vaccine.

In embodiments, the tumor-specific antigens (novopeptides) disclosedherein can come from or relate to any known tumor cell. Thuscontemplated herein are methods of screening for tumor specificantigens, wherein the tumor cell is from a cancer selected from thegroup of cancers consisting of lymphomas (Hodgkin's and non-Hodgkin's),B cell lymphoma, T cell lymphoma, leukemias, myeloid leukemia,carcinomas, carcinomas of solid tissues, squamous cell carcinomas,squamous cell carcinomas of the mouth, throat, larynx, and lung,adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas,neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,hypoxic tumours, myelomas, AIDS-related lymphomas or sarcomas,metastatic cancers, mycosis fungoides, bladder cancer, brain cancer,nervous system cancer, lung cancers such as small cell lung cancer andnon-small cell lung cancer, ovarian cancer, pancreatic cancer, prostatecancer, hepatic cancer, colon cancer, cervical cancer, cervicalcarcinoma, breast cancer, and epithelial cancer, renal cancer,genitourinary cancer, esophageal carcinoma, head and neck carcinoma,large bowel cancer, hematopoietic cancers, and testicular cancer. Thesource of novopeptides can be from sequences relating to any tumor type,and some novopeptides are applicable to a wide variety of tumors; whenpooled, an appropriate selection of such novopeptides can give rise to auniversal prophylactic vaccine.

An advantage of the disclosed approach is that it provides insights intocancer. For example, one of the 11-mer frameshift peptides that wasisolated (peptide FS 6-21) was found to have homology to a region ofHuntingtin interacting protein (HIP1), the level of which is positivelycorrelated with disease progression in patients with Huntington'sdisease (Kerr, 2002). Interestingly, there are a number of studies thathave shown that this disorder is associated with a significantly lowerincidence of cancer (Sorenson et al., 1999).

In embodiments, novopeptides are provided that are associated withcancer cells. The disclosed components can be used to prepare thedisclosed compositions as well as in the compositions themselves to beused within the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed, whilespecific reference of each various individual and collectivecombinations and permutations of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular novopeptide or novopeptide associated mutationor variation (e.g., peptide FS 1-78, peptide FS 6-21, or peptide FSSMC1A) is disclosed and discussed and a number of modifications that canbe made to a number of molecules including the FS 1-78, FS 6-21, and FSSMC1A are discussed, specifically contemplated is each and everycombination and permutation of FS1-78, FS 6-21, and FS SMC1A and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

The disclosed screening methods can be used to identify novopeptideassociated mutations or variations associated with cancers. Thedisclosed novopeptide associated mutations or variations aredifferentially expressed in cancerous cells as compared to noncancerouscells. Since novopeptide associated mutations or variations occur in allcancers tested, the novopeptides furnish a basis for therapeuticvaccines. Therefore, disclosed herein are vaccines for a cancercomprising one or more novopeptides, wherein the novopeptide is derivedfrom a novopeptide associated mutation or variation, and wherein thenovopeptide(s) is identified via the disclosed screening methods or byany other method. Specifically disclosed herein are novopeptides,wherein the novopeptide is associated with a frameshift of the SMC1Agene. Disclosed herein, are frameshift mutation peptides that have beenidentified that are present only or predominantly in cancerous tissue.See, for example, the list of peptides in the Sequence Listing.Specifically disclosed herein are tumor-specific antigens, wherein theantigen is a peptide as set forth in SEQ ID NOs: 2, 4, 6, and 8. It isunderstood that there are numerous nucleotide sequences that can encodefor the peptides disclosed herein. For example, one example of anucleotide that encodes the peptide set forth in SEQ ID NOs: 2, 4, and 6are the nucleotide sequences of SEQ ID NOs: 1, 3, and 5, respectively.It is understood and herein contemplated are each and every nucleotidesequence that encodes the disclosed peptides.

Because the novopeptides in the Sequence Listing have been shown by thepresent screening method to be present only or predominantly innon-normal (e.g., cancerous) tissue, each disclosed novopeptide can beused as a reagent for detecting the presence of anti-novopeptideantibodies in a subject. Thus, the novopeptides have utility in a methodof detecting the presence of non-normal (e.g., cancerous) tissue in asubject as further described below.

Because the novopeptide associated mutation or variation is presentand/or expressed at higher levels in cancerous tissue as compared tonormal or noncancerous tissue, the novopeptide associated mutations orvariations itself can be used as the basis for a target for drug orantibody treatment as well as methods of identifying subjects at riskfor a cancer by virtue of the presence of the novopeptide associatedmutation or variation. Therefore, the disclosure hereof extends toantibodies to novopeptides or to FS-novopeptides or to non-MSnovopeptides. It is understood that the antibody can be specific to anynovopeptide disclosed herein. For example, the antibody can be directedto a frameshift mutant of the SMC1A gene. The disclosure hereof extends,by way of example only, to antibodies directed toward a novopeptidecomprising a sequence set forth in SEQ ID NOs: 2, 4, 6, or 8. It isunderstood that the antibody can be administered by itself or as acomponent of another composition. Thus, herein disclosed arecompositions comprising antibodies specific for the tumor specificantigens disclosed herein. The vaccines disclosed herein can be used totreat or prevent cancer due to the presence of the novopeptides ornovopeptide associated mutations or variations in tumor cells.Alternatively, since the mutation does not occur or is not prevalent innormal cells it can also be used as a prophylactic vaccine. Thusdisclosed herein are compositions comprising a prophylactic vaccineincluding components as disclosed herein such that they would bepredicted to provide protection to 10% or more of a population against aparticular tumor or group of tumors by taking into account the frequencyof the peptides in the tumors and the frequency of the MHCI types in thepopulation.

Thus, disclosed herein are embodiments of methods of treating cancercomprising administering to a subject in need thereof the vaccinesdisclosed herein. Also disclosed herein are embodiments of methods ofpreventing a cancer comprising administering to a subject at riskthereof the vaccines disclosed herein. The disclosed vaccines can beused to treat cancer due to the presence of disclosed tumor-specificantigens in all cancers. Contemplated and disclosed herein arevaccinations and compositions for treating or preventing cancer whereinthe cancer is of any type, including, by way of example only, thoseselected from the group of cancers consisting of lymphomas (Hodgkin'sand non-Hodgkin's), B cell lymphoma, T cell lymphoma, leukemias, myeloidleukemia, carcinomas, carcinomas of solid tissues, squamous cellcarcinomas, squamous cell carcinomas of the mouth, throat, larynx, andlung, adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas,neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,hypoxic tumours, myelomas, AIDS-related lymphomas or sarcomas,metastatic cancers, mycosis fungoides, bladder cancer, brain cancer,nervous system cancer, lung cancers such as small cell lung cancer andnon-small cell lung cancer, ovarian cancer, pancreatic cancer, prostatecancer, hepatic cancer, colon cancer, cervical cancer, cervicalcarcinoma, breast cancer, and epithelial cancer, renal cancer,genitourinary cancer, esophageal carcinoma, head and neck carcinoma,large bowel cancer, hematopoietic cancers, and testicular cancer.

The antibodies disclosed herein can be combined with other agents,molecules, or compounds to increase binding, elicit additional immuneresponses, or deliver toxic effects to the proximity of the targetantigen, e.g., to cells that express the frameshift mutation. Suchcombinations can occur through the formation of fusion constructs,immunoconjugates, or other combination platforms as known in the art.Thus, in embodiments, the antibodies disclosed herein can be combinedwith a toxin such as diphtheria toxin, ricin toxin, tetanus toxoid,botulinum toxin, or any other toxin as a fusion construct to form anantibody-toxin fusion. For example, the antibody-toxin fusion constructcan comprise the disclosed antibody fused to a diphtheria toxin. Inembodiments, the disclosed toxins such as tetanus and diphtheria cancomprise truncation mutants to avoid the antibody response from previousexposure to the toxin. For example, a diphtheria toxin can comprise atruncation mutant diphtheria toxin wherein the toxin comprises a 145-152amino acid truncation of the c-terminal end of the diphtheria toxin.

Immunogenic fusion protein derivatives, such as those described in theexamples, may be made by fusing a polypeptide sufficiently large toimprove immunogenicity to the target sequence by cross-linking in vitroor by recombinant cell culture transformed with DNA encoding the fusion.For example, a FS novopeptide can be fused to a carrier such as aprotein or sugar. Methods for improving the immunogenic properties of apeptide by fusing, conjugating or otherwise associating it with a haptenor other carrier are well known to persons having ordinary skill in theart of immunology.

The terms homology, similarity, and identity may be used interchangeablyherein and have equivalent meaning except where otherwise specificallyindicated or where context requires otherwise. Thus, for example, if theuse of the word homology is used between two non-natural sequences it isunderstood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather refers to thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. In general, variants ofgenes and proteins herein disclosed typically have at least, about 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology orsequence identity to the stated sequence or the native sequence or othersequence with which comparison is made. Those of skill in the artreadily understand how to determine the homology of two proteins ornucleic acids, such as genes. For example, the homology can becalculated after aligning the two sequences so that the homology is atits highest level.

In embodiments, homology can be computed using published algorithms.Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, WI), or by inspection.

Homology can be determined for nucleic acids by any of the methods knownto a person having ordinary skill in the art, including withoutlimitation, for example, the algorithms disclosed in Zuker, M. Science244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710,1989, Jaeger et al Methods Enzymol. 183:281-306, 1989 which are hereinincorporated by reference for at least the material related to nucleicacid alignment. In embodiments, any of the methods typically can be usedand in certain instances the results of these various methods maydiffer, but the skilled artisan understands that if identity is foundwith at least one of these methods, the sequences would be said to havethe stated identity, and be disclosed herein.

For example, as used herein, a sequence may be recited as having aparticular percent homology to another sequence when the sequences havethe recited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

A variety of molecules disclosed herein that are nucleic acid based,including for example the nucleic acids that encode FS1-78, FS 6-21, FSSMC1A, or fragments thereof, as well as various functional nucleicacids. In embodiments, the disclosed nucleic acids may be made up of,for example, nucleotides, nucleotide analogs, or nucleotide substitutes.Non-limiting examples of these and other molecules are discussed herein.For example, when a vector is expressed in a cell, the expressed mRNAwill typically be made up of A, C, G, and U. The disclosure hereofextends to the nucleic acid sequences described herein and to any andall other nucleic acids that are similar or homologous thereto,regardless of whether comprised in whole or in part of nucleotides,nucleotide analogs, or nucleotide substitutes, or any combinationthereof and regardless of whether or not linked to conjugates or othermolecules or moieties. Likewise, if, for example, an antisense moleculeis introduced into a cell or cell environment through exogenousdelivery, it is advantageous that the antisense molecule be made up ofnucleotide analogs that reduce the degradation of the antisense moleculein the cellular environment.

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Anon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate). There are manyvarieties of these types of molecules available in the art and availableherein.

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties. There are many varieties of these typesof molecules available in the art and available herein.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid. There are many varieties of these types of molecules available inthe art and available herein.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of thesetypes of molecules available in the art and available herein.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or 0) at the C6 position of purinenucleotides.

There are a variety of sequences related to the protein and/or peptidemolecules disclosed herein, including without limitation and by way ofexample only, FS 1-78 (SEQ ID NO: 2), FS 6-21, and FS SMC1A, or any ofthe nucleic acids disclosed herein, including without limitation and byway of example only, those encoding all or part of FS 1-78, FS 6-21, andFS SMC1A. The disclosure hereof extends to analogs of these genes, aswell as other alleles of these genes, and splice variants and othertypes of variants, in humans and in any other species exhibitingspecific immunity including without limitation mammals, fish, and birds.The sequences of various of the foregoing to the extent currently knownare available in a variety of protein and gene databases, includingGenbank. Such sequences available at the time of filing this applicationat Genbank are herein incorporated by reference in their entireties aswell as for individual subsequences contained therein. Genbank can beaccessed via the web site of the National Center for BiotechnologyInformation. Those of skill in the art understand how to resolvesequence discrepancies and differences and to adjust the compositionsand methods relating to a particular sequence and to other relatedsequences. Primers and/or probes can be designed for any given sequencegiven the information disclosed herein and known in the art.

As discussed herein there are numerous variants of the FS1-78, FS 6-21and FS SMC1A protein that are known and herein contemplated. In additionto the known functional FS 1-78, FS 6-21, FS SMC1A, and othernovopeptide variants there are derivatives of the FS1-78, FS6-2, and FSSMC1A and other novopeptides which also function in the disclosedmethods and compositions. Protein and peptide variants and derivativesare well understood to those of skill in the art and can involve aminoacid sequence modifications. For example, amino acid sequencemodifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to ten residues.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. These variants may be prepared bysite specific mutagenesis of nucleotides in the DNA encoding theprotein, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture, or by any of the othermethods known to a person having ordinary skill in the art for making orobtaining proteins or peptides having a specified sequence. Techniquesfor making substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example M13 primer mutagenesis andPCR mutagenesis. Amino acid substitutions are typically of singleresidues, but can occur at a number of different locations at once;insertions usually will be on the order of about from 1 to 10 amino acidresidues; and deletions will range about from 1 to 30 residues.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final construct. Mutations to DNA encoding thevariant should typically not place the sequence out of reading frame andpreferably will not create complementary regions that could produceundesired secondary mRNA structure. Substitutional variants are those inwhich at least one residue has been removed and a different residueinserted in its place. Substitutions in which a residue is replaced witha different amino acid having chemical, biological, or other propertiessimilar to those of the residue that it is replacing, such as, forexample, those shown in the following tabulation, are referred to asconservative substitutions.

Conservative Amino Acid Abbreviation Substitution alanine ala A serallosoleucine alle arginine arg R lys, gln asparagine asn N gln, hisaspartic acid asp D glu cysteine cys C ser glutamic acid glu E asnglutamine gin Q asp glycine gly G pro histidine his H asn, glnisoleucine ile I leu, val leucine leu L ile, val lysine lys K arg, glnphenylalanine phe F leu, ile proline pro P met, leu, tyr pyroglutamicacid pyr serine ser S thr threonine thr T ser tyrosine tyr Y tyrtryptophan trp W trp, phe valine val V ile, leu

The replacement of one amino acid residue with another that isbiologically and/or chemically similar is known to those skilled in theart as a conservative substitution. Without limiting the generality ofthe foregoing, and by way of example only, the substitutions tabulatedabove are conservative substitutions. In embodiments, conservativesubstitutions may also include any substitution that would be regardedby one having ordinary skill in the art as conservative, and mayinclude, without limitation, substitutions having a log odds score ofzero or above in the BLOSUM 62 matrix, or having a relatively high logodds score in any other substitution matrix in common usage. Forexample, a conservative substitution may entail replacing onehydrophobic residue for another, or one polar residue for another. Suchsubstitutions include combinations such as, for example, Gly, Ala; Val,Ile, Leu; Asp, GIu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Suchconservatively substituted variations of each explicitly disclosedsequence are included within the polypeptides disclosed herein.

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shownabove, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine; or (e) by increasing the number of sites for sulfationand/or glycosylation.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or 0-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 2 sets forth a particular sequence of peptide1-78 and SEQ ID NO:4 sets forth a particular sequence of peptide 6-21.Specifically disclosed are variants of these and other proteins hereindisclosed which have at least 70% or 75% or 80% or 85% or 90% or 95%homology to the stated sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins. For example,the homology can be calculated after aligning the two sequences so thatthe homology is at its highest level. It is understood that thedescription of conservative mutations and homology can be combinedtogether in any combination, such as embodiments that have at least 70%homology to a particular sequence wherein the variants are conservativemutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the proteinsequence set forth in SEQ ID NO:2 is set forth in SEQ ID NO:l. Inaddition, nucleic acid sequences encoding disclosed conservativederivatives of SEQ ID NO:2 are also disclosed. It is also understoodthat while no amino acid sequence indicates what particular DNA sequenceencodes that protein within an organism, where particular variants of adisclosed protein or peptide are disclosed herein, the known nucleicacid sequence that encodes that protein is also known and hereindisclosed and described.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent than the 20 naturally occurring amino acidstabulated above. The opposite stereoisomers of naturally occurringpeptides are disclosed, as well as the stereoisomers of peptide analogs.These analog amino acids can readily be incorporated into polypeptidechains by charging tRNA molecules with the amino acid of choice andengineering genetic constructs that utilize, for example, amber codons,to insert the analog amino acid into a peptide chain in a site specificway (Thorson et al., Methods in Molec. Biol. 11:A2>-12> (1991), Zoller,Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology& Genetic Engineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Biotechnology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH2NH—, —CH2S—, —CH2-CH2-,—CH═CH-(cis and trans), —COCH2-, —CH(OH)CH2-, and —CHH2SO—. (These andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (Mar. 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH2NH—, CH2CH2-); Spatola et al. Life Sci38:1243-1249 (1986) (—CH H2-S); Hann J. Chem. Soc Perkin Trans. 1307-314(1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH2-); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH2-); Szelke et al. European Appin, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH2-); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH2-); and Hruby V., Sci 31:189-199 (1982)(—CH2-S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH2NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and peptide analogs may have enhanced or desirableproperties, such as, more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others.

D-amino acids can be used to generate more stable peptides, becauseD-amino acids are not recognized by peptidases. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) can be used togenerate more stable peptides. Cysteine residues can be used to cyclizeor attach two or more peptides together. This can be beneficial toconstrain peptides into particular conformations. (Rizo and GieraschAnn. Rev. Biochem. 61:387 (1992), incorporated herein by reference).

The compositions disclosed herein can be used for treatment orprophylaxis against any disease where uncontrolled cellularproliferation occurs, such as cancers. It is understood and hereincontemplated that, in embodiments, such compositions may becharacterized by any novopeptide associated mutation or variationdisclosed herein. Therefore, disclosed herein are embodiments of methodsof treating a cancer comprising administering a composition to a subjectin need thereof, wherein the composition comprises a novopeptide, aFS-novopeptide, a non-MS novopeptide, a non-MS novopeptide that is alsoa FS-novopeptide, and/or any combination of the foregoing. Thus, forexample, the novopeptide associated mutation or variation can be aframeshift of the SMC1A gene. It is also understood that the frameshiftcan be a peptide. Thus, by way of example only and without limiting thegenerality of the foregoing, disclosed herein are embodiments of methodsof treating cancer comprising administering to a subject in need thereofa composition comprising a novopeptide, wherein the novopeptidecomprises the sequence set forth in SEQ ID NO: 2, 4, 6, or 8.

An aspect of the disclosure hereof relates to the identification ofnovopeptides, including, for example, novopeptides referred to herein asnovopeptide vaccine antigens, suitable for inclusion in a prophylacticor therapeutic cancer vaccine. A novopeptide vaccine antigen is anovopeptide that is capable, when administered in an appropriatelyconstituted prophylactic or therapeutic vaccine, of fostering anappreciable immune response, which may be humoral, cellular, or both,against at least one cancerous cell type in at least one individual.

The utility of the methods, compositions, and articles of manufacturedisclosed herein has been convincingly demonstrated in an animal model,as disclosed herein in the examples, and selected aspects of thedisclosed technology capable of being demonstrated without a need forhuman clinical testing have been experimentally confirmed in otherexperiments disclosed herein. The disclosure hereof is applicable tohuman and murine cancers, and similarly to cancers of all otherorganisms having mechanisms for specific immunity, specificallyincluding all mammals, as well as birds and fish. The disclosure hereofis of high potential significance not only in providing prophylactic andtherapeutic interventions for human cancer, but also for its veterinaryapplications, particularly in companion animals such as dogs and cats,for which cancer is a leading cause of death. Embodiments disclosedherein can readily be applied to dog cancer since the dog genomesequence determination has been completed.

While significant progress has been made over the past decade inunderstanding the basic immunology underlying cancer, thus far no onehas produced a cancer vaccine that can, reliably and consistently,induce tumor destruction or improve patient survival (Lewis, 2004; Leaf,2004). The scientific literature discloses a variety of cancervaccination strategies that have been investigated by others, eachproving less than ideal. The majority of personalized cancer vaccinestudies to date have focused on the use of undefined whole tumor-cellextracts prepared from a patient's own tumor. Experiments usingautologous vaccines in melanoma have shown that, in principle,immunologic intervention can enhance specific anti-tumor immuneresponses, and even mediate regression in some cases, but this approachpresents difficult challenges, including (1) the potential for causingautoimmunity; (2) dilution of TAAs since the majority of antigens willbe “normal”; (3) undermining of specificity (one of the most attractiveunique features of immunotherapy); (4) dependence on the patient havinga large enough tumor to make the vaccine, precluding early treatment;and (5) the need for custom preparation of a personalized vaccine foreach patient.

Another approach to cancer vaccination is to use vaccine formulationscomposed of known and defined TAAs, since this maximizes specificity andobviates the problem of antigen dilution. To date, several hundred humanTAAs have been identified using a variety of strategies, and criteriaexist for selecting TAAs suitable for immunotherapy. Functionally, TAAsmay be classified as self and non-self. Self-TAAs are derived fromnon-mutated genes whose expression is limited to certain tissues or toover-expressed proteins. Most TAAs identified and tested to date areself antigens. Potential problems associated with such antigens includeautoimmunity and tolerance. For practical purposes, this limits the useof self TAAs to non-vital organs (such as reproductive organs).Pre-existing immune tolerance to self antigens is also problematic, fornot only does it suppress a desired anti-tumor immune response, but morerecently it has emerged as a possible mechanism of immune escape.

All or most TSAs are non-self antigens and can originate eitherexogenously (such as those derived from viral proteins invirally-associated tumors, e.g. human papilloma virus) or endogenously.The latter subclass includes un-mutated proteins that might never havebeen presented to the immune system before (some embryonic or immuneprivileged antigens), as well as mutated proteins that arise as aconsequence of mutations in tumors. Mutation-derived TSAs can arise fromevents such as point mutations, frame shift mutations, translocations,improper splicing, and post transcriptional events. TSAs have a greatadvantage over self TAAs as cancer vaccines since they avoid theproblems of autoimmunity and systemic tolerance. In mouse models TSAshave been shown to generate high-avidity T cell responses more readilythan self TAAs.

In principle, vaccination can be used either prophylactically ortherapeutically. As a practical matter, therapeutic vaccinationstrategies face several difficult challenges, and, in general, havefailed to fulfill their early promise. Many or most antigens presentedby cells in an established tumor are recognized as self by the immunesystem. To the extent that tumor cells do display mutated antigens thatthe immune system is capable of recognizing as non-self, by the timetumor development has advanced sufficiently to allow diagnosis, immunetolerance to the mutated antigens will have developed owing to theirgradual exposure to the immune system in the absence of theco-regulatory danger signals that would be required for an immuneresponse. Recent studies have shown that in the absence ofco-stimulatory signals, tolerance can be induced even to foreignantigens expressed by a tumor. MHC expression is often down-regulated orimpaired in established tumor cells, reducing the display of anynon-self antigen, and the reduced MHC expression is of course selectedfor to the extent that any therapeutic immunization strategy iseffective in killing cells that do display recognizable non-self antigenin MHC. (The term “MHC” is used herein in a generic sense and isintended to include MHC, HLA, and any other entities at least one ofwhose functions is to display endogenous or exogenous antigens orfragments thereof on the surface of a cell. Where reference is made to aparticular MHC class, the reference includes any corresponding class ofHLA or other such entity.) Finally, it has been shown by multiple groupsthat immunization with irradiated tumor cells, tumor cell lysate, ortumor-derived heat shock proteins (HSPs) protects only against challengewith the same tumor; it does not protect against challenge with adifferent tumor; vaccines derived from one tumor did not protect againstanother. These findings have led to an assumption nearly universallyheld in the field of cancer immunology, but disproved by theexperimental evidence disclosed herein, that cancer vaccines must bepersonalized. Companies exist based on a technology in which theyreceive a tumor from a patient, isolate HSPs or extracts from thattumor, and return the tumor derived HSPs or extracts as apatient-specific vaccine. Early clinical trials implementing thisapproach showed promise, but it is expensive and not every cancerpatient has enough tumor from which to make a vaccine. A recent PhaseIII trial by Antigenics, Inc. using this strategy was stopped for lackof efficacy.

A fundamental problem for prophylactic vaccination as a cancerpreventative treatment has been the supposition that each tumor in eachorganism presents a unique immunological profile, and the consequentassumption that no prophylactic vaccine could offer a practicablebreadth of protection against multiple tumor types or even againstmultiple variants of a single tumor type. The problem is exacerbated bythe assumed need for any vaccine to be personalized to the organismreceiving it. In contrast, a basic contention hereof is that there arenovopeptides that are produced in common between two or more types ofcancers and that these can be used to formulate a prophylactic vaccine.The challenge was to develop a systematic method to find suchnovopeptides; such a method is disclosed herein.

The peptides disclosed herein can be administered to a subject as apeptide or encoded by a nucleic acid. Thus, for example, disclosedherein are embodiments of methods of treating a cancer comprisingadministering a composition to a subject in need thereof, wherein thecomposition comprises a tumor-specific antigen, and wherein thetumor-specific antigen is a novopeptide, a FS-novopeptide, a non-MSnovopeptide, a non-MS novopeptide that is also a FS-novopeptide , and/orany combination of the foregoing, and/or wherein the tumor-specificantigen is a peptide encoded by a nucleic acid set forth in SEQ ID NOs:1, 3, or 5. The nucleic acids encoding the novopeptides disclosed hereincan be provided by any gene delivery system disclosed herein such asgene gun, viral vector, or plasmid.

“Treatment” means a method of reducing the effects of a disease orcondition. Treatment can also refer to a method of reducing the diseaseor condition itself rather than just the symptoms. The treatment can beany reduction from native levels and can be but is not limited to thecomplete ablation of the disease, condition, or the symptoms of thedisease or condition. For example, a disclosed method for reducing theeffects of a cancer is considered to be a treatment if there is a 10%reduction in one or more symptoms of the disease (e.g., tumor size) in asubject with the disease when compared to native levels in the samesubject or control subjects. Thus, the reduction can be a 10, 20, 30,40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between ascompared to native or control levels. It is also understood andcontemplated herein that treatment can refer to any reduction in theprogression of a disease or cancer. Thus, for example, a method ofreducing the effects of a cancer is considered to be a treatment ifthere is a 10% reduction in the tumor growth rate relative to a controlsubject or tumor growth rates in the same subject prior to thetreatment. It is understood that the reduction can be a 10, 20, 30, 40,50, 60, 70, 80, 90, 100%, or any amount of reduction in between ascompared to native or control levels.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity,response, condition, disease, or other biological parameter. This caninclude but is not limited to the complete ablation of the activity,response, condition, or disease. This may also include, for example, a10% reduction in the activity, response, condition, or disease ascompared to the native or control level. Thus, the reduction can be a10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

The disclosed methods can be used for the treatment or inhibition of anycancer. Thus disclosed herein are embodiments of methods of treating,preventing, or inhibiting cancer, wherein the cancer may be of any type,such as, by way of example only, a cancer selected from the group ofcancers consisting of lymphomas (Hodgkin's and non-Hodgkin's), B celllymphoma, T cell lymphoma, leukemias, myeloid leukemia, carcinomas,carcinomas of solid tissues, squamous cell carcinomas, squamous cellcarcinomas of the mouth, throat, larynx, and lung, adenocarcinomas,sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas,plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumours,myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers,mycosis fungoides, bladder cancer, brain cancer, nervous system cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, ovarian cancer, pancreatic cancer, prostate cancer, hepaticcancer, colon cancer, cervical cancer, cervical carcinoma, breastcancer, and epithelial cancer, renal cancer, genitourinary cancer,esophageal carcinoma, head and neck carcinoma, large bowel cancer,hematopoietic cancers, and testicular cancer.

It is understood that, in addition to the present methods of identifyinga subject at risk of developing cancer, the identification of subjectsat risk of developing a cancer can be accomplished by any means known inthe art. Thus, for example, a subject at risk can be identified byexposure to a known carcinogen, behavioral activities associated withcancer (e.g., smoking with respect to lung cancer), or geneticpredisposition to a given cancer. Specifically disclosed herein areembodiments of methods of preventing a cancer in a subject at riskthereof wherein the subject is identified by genetic screening. Becausethe frameshift peptides disclosed herein are associated with cancer, thepresence of the frameshift can be used to identify subjects at risk ofdeveloping a cancer. Therefore, disclosed herein are embodiments ofmethods of identifying a subject at risk for developing a cancercomprising obtaining a tissue sample from the subject and contacting anantibody with the tissue sample, wherein antibody binding indicates thesubject is at risk for the cancer.

Compounds disclosed herein may also be used for the treatment ofprecancer conditions such as cervical and anal dysplasias, otherdysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, andneoplasias.

The disclosed methods can be used to treat or protect any subject inneed thereof or at risk of acquiring any disease disclosed herein. Inembodiments, a subject may include any animal capable of displayingspecific immunity such as bird, fish, and mammal. Thus, for example, asubject for use with any of the disclosed methods can be human,chimpanzee (or other non-human primate), monkey, cow, horse, pig, dog,cat, rat, guinea pig, and mouse.

A significant advantage of the methods, compositions, and articles ofmanufacture disclosed herein is that vaccination with a singlenovopeptide has been shown capable of conferring immunoprotectionagainst more than one tumor type and in unrelated individuals, asdemonstrated by the examples disclosed herein. This is a highly novelresult, particularly in the light of the widely held dogma based on thewhole-cell vaccine studies previously noted that immunization with onetumor cell line does not cross-protect against another. The resultsshown here may be reconciled with the whole-cell vaccine studies byobserving that tumors do have antigens in common, but immunization withcell lysates or irradiated tumors do not show cross-protection becausethe concentration of cross protective peptides in MHC is not high enoughin whole-cell vaccines to activate T cells; in other words, whole-cellvaccine strategies fail because of antigen dilution. Prevaccination withone or a few novopeptides concentrates the immune system on theseantigens and confers protection. This experimental finding leads to thevery important result that novopeptides expressed in common by multipletumor types can support prophylactic vaccination conferringimmunoprotection against those tumor types. This is an importantconceptual, experimental, and practical breakthrough. It will be notedthat the disclosure hereof provides an effective and systematic methodfor finding and evaluating novopeptides that are commonly expressedamong multiple tumor types.

In some cases a novopeptide produced in human tumors will be the same orvery similar to that produced in an animal tumor model such as mouse ordog. For example, the FS 1-78 and FS 6-21 novopeptides described belowwere found in mouse tumors as there described, but have also beenidentified in certain human tumors. The SMC1A novopeptide, describedbelow, was found originally by searching human databases, but also isexpressed in mouse tumors. If a novopeptide is found in both human andmouse tumors, significant evidence of the potential effectiveness of thenovopeptide as a tumor vaccine antigen in humans can be obtained byimmunizing mice and challenging with the appropriate tumor line.Alternatively, cancer prone mice can be vaccinated with the novopeptideto determine whether tumorigenesis and/or tumor progression is reducedor eliminated.

Disclosed herein are therapeutic antibodies to a tumor-specific antigen,wherein the antigen is a novopeptide identified by the steps comprisingidentifying a novopeptide by informatics, genomics, proteomics, orimmunological screens; and determining that the novopeptide induces animmune response that differentiates between tumor cells and normalcells. It is understood and herein contemplated that the novopeptide canbe a tumor-specific antigen. Thus, for example, the novopeptide cancomprise the sequence set forth in SEQ ID NO: 2, 3, or 6. Similarly, thenovopeptide can comprise a frameshift of the SMC1A gene. Thus forexample, the novopeptide can comprise the sequence set forth in SEQ IDNO: 8. It is also understood that the disclosed therapeutic antibodiescan be used alone or in combination with another agent as a therapeutictreatment. It is also contemplated herein that the therapeutictreatments disclosed herein can be used to treat cancer. In other words,disclosed herein are embodiments of methods of treating a cancercomprising administering to a subject the therapeutic antibodiesdisclosed herein or identified by the methods disclosed herein. Thus,for example, disclosed herein are embodiments of methods of therapeutictreatment, wherein the cancer may be of any type, such as, by way ofexample only, a cancer selected from the group of cancers consisting oflymphomas (Hodgkin's and non-Hodgkin's), B cell lymphoma, T celllymphoma, leukemias, myeloid leukemia, carcinomas, carcinomas of solidtissues, squamous cell carcinomas, squamous cell carcinomas of themouth, throat, larynx, and lung, adenocarcinomas, sarcomas, gliomas,high grade gliomas, blastomas, neuroblastomas, plasmacytomas,histiocytomas, melanomas, adenomas, hypoxic tumours, myelomas,AIDS-related lymphomas or sarcomas, metastatic cancers, mycosisfungoides, bladder cancer, brain cancer, nervous system cancer, lungcancers such as small cell lung cancer and non-small cell lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, hepatic cancer,colon cancer, cervical cancer, cervical carcinoma, breast cancer, andepithelial cancer, renal cancer, genitourinary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers, and testicular cancer.

In embodiments, the compositions disclosed herein can be used forexample as targets in combinatorial chemistry protocols or otherscreening protocols to isolate molecules that possess desired functionalproperties related to inhibiting tumor growth and treating cancer. Thus,disclosed herein are embodiments of methods of screening for a cancertherapeutic or prophylactic comprising contacting the candidatetherapeutic or prophylactic with a novopeptide, wherein a candidatetherapeutic or prophylactic that binds the novopeptide is selected forfurther evaluation as a therapeutic or prophylactic.

The disclosed compositions can also be used as diagnostic tools relatedto diseases such as cancer. For example, the disclosed methods can beused to determine if a cell growth is cancerous. Thus, disclosed hereinare embodiments of methods of diagnosing a tumor or other growth ascancerous or precancerous comprising screening for a novopeptidecomprising obtaining a tumor cell, extracting RNA from the cell, andassaying for novopeptide associated mutations or variations, wherein thepresence of a novopeptide associated mutation or variation indicates thetumor is cancerous or potentially cancerous. Also disclosed are methodsof diagnosing an individual with cancer comprising obtaining a tissuesample, and screening for the presence of a novopeptide associatedmutation or variation. It is understood that the tissue can be anytissue present in the subject. For example, the tissue can be blood,saliva, skin, or cells from a tissue biopsy. It is also understood thatthe disclosed tissues can be obtained by any method known in the artsuch as, for example, lung lavage, venous bleeding, tissue biopsy, ormucosal tissue swab. Thus, for example, disclosed herein are embodimentsof methods of diagnosing wherein the sample is blood. The method caninvolve determining the presence of a novopeptide associated mutation orvariation identified to be associated with cancer. Alternatively, themethod can involve screening for the presence of an immune response to anovopeptide. It is understood that the immune response can be anantibody or cell-mediated response. Thus, for example, the immuneresponse can be a T cell response such as a CD8 T cell response (e.g.,cytolytic killing or cytokine secretion) or CD4 T cell response(cytokine secretion). It is specifically contemplated herein that anyknown immunological measure may be used to determine the presence of theimmune response. For example, antibody responses can be measured byELISA, ELISPOT, or agglutination assays. T cell responses can bedetected by, for example, ELISA, ELISPOT, tetramer staining,intracellular cytokine staining, or chromium release assays.

It is possible that novopeptide associated mutations or variationsidentified by the methods disclosed herein may result in an otherwisenon-oncogenic gene becoming oncogenic. For example, the SMC1A gene isnot oncogenic; however, a frameshift of the SMC1A gene as disclosedherein may be oncogenic. The methods of detecting novopeptide associatedmutations or variations in tumor cells disclosed herein showed aframeshift in the SMC 1A gene which as a frameshift mutant is oncogenic.Thus, disclosed herein are embodiments of methods of identifyingoncogenes, comprising detecting a novopeptide associated mutation orvariation in a gene not previously associated with cancer.

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

For example, nucleic acids, such as, for example, oligonucleotides to beused as primers or nucleic acids encoding novopeptides, can be madeusing standard chemical synthesis methods or can be produced usingenzymatic methods or any other known method. Such methods can range fromstandard enzymatic digestion followed by nucleotide fragment isolation(see for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, forexample, by the cyanoethyl phosphoramidite method using a Milligen orBeckman System 1Plus DNA synthesizer (for example, Model 8700 automatedsynthesizer of Milligen-Biosearch, Burlington, Mass. or ABI Model 380B).Synthetic methods useful for making oligonucleotides are also describedby Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriesterand phosphite-triester methods), and Narang et al., Methods EnzymoL,65:610-620 (1980), (phosphotriester method). Protein nucleic acidmolecules can be made using known methods such as those described byNielsen et al., Bioconjug. Chem. 5:3-7 (1994).

An example of a method of producing the disclosed proteins and peptidesis to link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(Xert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the disclosed proteins, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a peptide or protein canbe synthesized and subsequently cleaved from the resin, thereby exposinga terminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form an antibody, or fragment thereof, or otherpolypeptide of interest. (Grant G A (1992) Synthetic Peptides: A UserGuide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed.(1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY (whichis herein incorporated by reference at least for material related topeptide synthesis). Alternatively, a peptide or polypeptide may beindependently synthesized in vivo as described herein. Once isolated,these independent peptides or polypeptides may be linked to form apeptide or fragment thereof via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments may be chemically linkedwhere the bond formed between the peptide segments as a result of thechemical ligation is an unnatural (nonpeptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof.These may be chosen for their ability to interact with FS 1-78, FS 6-21,FS SMC1A, or other novopeptides or targets of interest, and inembodiments may be used such that tumor growth is inhibited. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic and/or prophylactic activities may be tested accordingto known clinical testing methods.

As used herein, the term “antibody” encompasses, but is not limited to,whole immunoglobulin (i.e., an intact antibody) of any class. Nativeantibodies are usually heterotetrameric glycoproteins, composed of twoidentical light (L) chains and two identical heavy (H) chains.Typically, each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (V(H)) followed by a numberof constant domains. Each light chain has a variable domain at one end(V(L)) and a constant domain at its other end; the constant domain ofthe light chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Particular amino acid residues are believedto form an interface between the light and heavy chain variable domains.The light chains of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (k) andlambda (1), based on the amino acid sequences of their constant domains.Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of human immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-I, IgG-2, IgG-3, and IgG-4; IgA-I and IgA-2. Oneskilled in the art would recognize the comparable classes for mouse. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively.

The term “variable” is used herein to describe certain portions of thevariable domains that differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a beta sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the beta sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat E. A.et al., “Sequences of Proteins of Immunological Interest,” NationalInstitutes of Health, Bethesda, Md.). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

As used herein, the term “antibody or fragments thereof encompasseschimeric antibodies and hybrid antibodies, with dual or multiple antigenor epitope specificities, and fragments, such as F(ab′)2, Fab′, Fab,sFv, scFv, and the like, including hybrid fragments. Thus, fragments ofthe antibodies that retain the ability to bind their specific antigensare provided. For example, fragments of antibodies which maintain FS1-78, FS 6-21, FS SMC1A binding activity are included within the meaningof the term “antibody or fragment thereof” Such antibodies and fragmentscan be made by techniques known in the art and can be screened forspecificity and activity according to the methods set forth in theExamples and in general methods for producing antibodies and screeningantibodies for specificity and activity (See Harlow and Lane.Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, NewYork, (1988)).

Also included within the meaning of “antibody or fragments thereof” areconjugates of antibody fragments and antigen binding proteins (singlechain antibodies) as described, for example, in U.S. Pat. No. 4,704,692,the contents of which are hereby incorporated by reference.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M J. Curr. Opin.Biotechnol. 3:348-354, 1992).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sd. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody, where context so indicates.Many non-human antibodies (e.g., those derived from mice, rats, orrabbits) are naturally antigenic in humans, and thus can give rise toundesirable immune responses when administered to humans. Therefore, theuse of human or humanized antibodies in the methods serves to lessen thechance that an antibody administered to a human will evoke anundesirable immune response.

In embodiments, human antibodies can be prepared using any operabletechnique. Examples of techniques for human monoclonal antibodyproduction include those described by Cole et al. (Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boemer et al. (J.Immunol., 147(1):86-95, 1991). Human antibodies (and fragments thereof)can also be produced using phage display libraries (Hoogenboom et al.,J. Mol. Biol, 227:381, 1991; Marks et al., J Mol. Biol, 222:581, 1991).

Human antibodies can also be obtained from transgenic animals. Forexample, transgenic, mutant mice that are capable of producing a fullrepertoire of human antibodies, in response to immunization, have beendescribed (see, e.g., Jakobovits et al., Proc. Natl Acad. ScL USA,90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, thehomozygous deletion of the antibody heavy chain joining region (J(H))gene in these chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production, and the successfultransfer of the human germ-line antibody gene array into such germ-linemutant mice results in the production of human antibodies upon antigenchallenge. Antibodies having the desired activity are selected usingEnv-CD4-co-receptor complexes as described herein.

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen), in some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human,in practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol, 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

Administration of the antibodies can be carried out as disclosed herein.Nucleic acid approaches for antibody delivery also exist. Broadlyneutralizing antibodies and antibody fragments can also be administeredto patients or subjects as a nucleic acid preparation (e.g., DNA or RNA)that encodes the antibody or antibody fragment, such that the patient'sor subject's own cells take up the nucleic acid and produce and secretethe encoded antibody or antibody fragment. The delivery of the nucleicacid can be by any operable means, such as, for example, those disclosedherein.

In embodiments, compositions disclosed herein can be used as targets forany combinatorial technique to identify molecules or macromolecularmolecules that interact with the disclosed compositions in a desiredway. Also disclosed are the compositions that are identified throughcombinatorial techniques or screening techniques in which thecompositions disclosed in SEQ ID NOS: 2, 4, 6, and 8 or portionsthereof, or other novopeptides disclosed herein or portions thereof, areused as the target in a combinatorial or screening protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecules, may be identified that have particular desiredproperties such as inhibition or stimulation of the target molecule'sfunction. The molecules identified and isolated when using the disclosedcompositions are also disclosed.

Combinatorial chemistry includes but is not limited to all art-acceptedmethods for isolating small molecules or macromolecules that are capableof binding either a small molecule or another macromolecule, typicallyin an iterative process. Proteins, oligonucleotides, and sugars areexamples of macromolecules. For example, oligonucleotide molecules witha given function, such as, for example, catalysis or ligand-binding, canbe isolated from a complex mixture of random oligonucleotides in whathas been referred to as “in vitro genetics” (Szostak, TIBS 19:89, 1992).One synthesizes a large pool of molecules bearing random and definedsequences and subjects that complex mixture, for example, approximately10¹⁵ individual sequences in 100 μg of a 100 nucleotide RNA, to someselection and enrichment process. Through repeated cycles of affinitychromatography and PCR amplification of the molecules bound to theligand on the column, Ellington and Szostak (1990) estimated that 1 in10¹⁰ RNA molecules folded in such a way as to bind a small molecule dye.DNA molecules with such ligand-binding behavior have been isolated aswell (Ellington and Szostak, 1992; Bock et al, 1992). Techniques aimedat similar goals exist for small organic molecules, proteins, antibodiesand other macromolecules known to those of skill in the art. Screeningsets of molecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material related to phage display andmethods relate to combinatorial chemistry)

A method for isolating proteins that have a given function is describedby Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc. Natl.Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorial chemistrymethod couples the functional power of proteins and the genetic power ofnucleic acids. An RNA molecule is generated in which a puromycinmolecule is covalently attached to the 3′ end of the RNA molecule. An invitro translation of this modified RNA molecule causes the correctprotein, encoded by the RNA to be translated. In addition, because ofthe attachment of the puromycin, a peptidyl acceptor which cannot beextended, the growing peptide chain is attached to the puromycin whichis attached to the RNA. Thus, the protein molecule is attached to thegenetic material that encodes it. Normal in vitro selection procedurescan now be employed to isolate functional peptides. Once the selectionprocedure for peptide function is complete traditional nucleic acidmanipulation procedures are performed to amplify the nucleic acid thatcodes for the selected functional peptides. After amplification of thegenetic material, new RNA is transcribed with puromycin at the 3′ end,new peptide is translated and another functional round of selection isperformed. Thus, protein selection can be performed in an iterativemanner similar to nucleic acid selection techniques. The peptide whichis translated is controlled by the sequence of the RNA attached to thepuromycin. This sequence can be anything from a random sequenceengineered for optimum translation (i.e. no stop codons etc.) or it canbe a degenerate sequence of a known RNA molecule to look for improved oraltered function of a known peptide. The conditions for nucleic acidamplification and in vitro translation are well known to those ofordinary skill in the art and may be performed as in Roberts and Szostak(Roberts R. W. and Szostak J. W. Proc. Natl. Acad. Sci. USA,94(23)12997-302 (1997)).

Another combinatorial method designed to isolate peptides is describedin Cohen et al. (Cohen B. A., et al., Proc. Natl. Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes and modifies two-hybridtechnology. Yeast two-hybrid systems are useful for the detection andanalysis of protein-protein interactions. The two-hybrid system,initially described in the yeast Saccharomyces cerevisiae, is a powerfulmolecular genetic technique for identifying new regulatory molecules,specific to the protein of interest (Fields and Song, Nature 340:245-6(1989)). Cohen et al., modified this technology so that novelinteractions between synthetic or engineered peptide sequences could beidentified which bind a molecule of choice. The benefit of this type oftechnology is that the selection is done in an intracellularenvironment. The method utilizes a library of peptide molecules thatattach to an acidic activation domain. For example, a peptide such as FS1-78 may be attached to a DNA binding domain of a transcriptionalactivation protein, such as Gal 4. By performing the two-hybridtechnique on this type of system, molecules that bind peptide FS 1-78,can be identified.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768 and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2, 3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721 ,099), polyketides(U.S. Pat. No. 5 ,712, 146), morpholino-subunits (U.S. Pat. No.5,698,685 and 5,506,337), sulfamides (U.S. Pat. No. 5,618,825), andbenzodiazepines (U.S. Pat. No. 5,288,514).

The disclosure hereof provides methods, compositions, and articles ofmanufacture useful in the formulation of prophylactic and/or therapeuticvaccines to be administered for the purpose of raising an immuneresponse against tumor cells. The disclosure hereof extends to thecomposition of novopeptide-based vaccines and to methods ofadministration thereof. A novopeptide-based vaccine may be prepared andadministered in any of the ways familiar to persons having ordinaryskill in the art, including the very simple approach of preparing avaccine comprising a novopeptide dissolved or suspended in a suitablecarrier, and administering it once or at predetermined intervals to theanimal or human patient to be vaccinated. However, success may also behad by other methods, and a particular approach entails geneticimmunization using gene gun technology, in which a vaccine isadministered in the form of a linear expression element encoding thedesired novopeptide, as illustrated in the examples below. Thecomposition of a vaccine may include both novopeptide and othercomponents. The inclusion of multiple distinct novopeptides may proveuseful in improving the level of immunoprotection conferred, and/or byconferring immunoprotection against additional tumor types, asillustrated by the examples below. In some embodiments, singlenovopeptides may be found to confer immunoprotection against more thanone tumor type, but the repertoire of target tumor types may also beexpanded by inclusion of additional novopeptides. The inclusion ofmultiple novopeptides is of particular utility in vaccines intended foradministration in humans, due to the desirability of including a numberand selection of novopeptides sufficient to ensure that at least onenovopeptide in the vaccine will be capable of being displayed by atleast one HLA type present in each individual in a predeterminedpercentage of the target population. In embodiments, two or morenovopeptides may be fused into a single entity; this is a standardpractice in the field of vaccine design. Novopeptide-based vaccines mayinclude other components familiar to a person having ordinary skill inthe art for improving the immunoprotection conferred or otherwiseimproving the efficacy and/or safety of the vaccine formulation,including without limitation and by way of example only, adjuvants andhapten carriers.

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as electroporation, lipofection, calcium phosphateprecipitation, plasmids, viral vectors, viral nucleic acids, phagenucleic acids, phages, cosmids, or via transfer of genetic material incells or carriers such as cationic liposomes. Appropriate means fortransfection, including viral vectors, chemical transfectants, orphysico-mechanical methods such as electroporation and direct diffusionof DNA, are described by, for example, in Wolff, J. A., et al., Science,247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991).Such methods are well known in the art and readily adaptable for usewith the compositions and methods described herein. In certain cases,the methods will be modified to specifically function with large DNAmolecules. Further, these methods can be used to target certain diseasesand cell populations by using the targeting characteristics of thecarrier.

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as peptide FS 1-78 into the cell withoutdegradation and include a promoter yielding expression of the gene inthe cells into which it is delivered. Viral vectors may include, forexample, Adenovirus, Adeno-associated virus, Herpes virus, Vacciniavirus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis andother RNA viruses, including these viruses with the HIV backbone. Alsopreferred are any viral families which share the properties of theseviruses which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of MMLV as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. A preferred embodiment is a viral vectorwhich has been engineered so as to suppress the immune response of thehost organism, elicited by the viral antigens. Preferred vectors of thistype will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase II transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promoter cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, L M., Retroviralvectors for gene transfer, in Microbiology-1985, American Society forMicrobiology, pp. 229-232, Washington, (1985), which is incorporated byreference herein. Examples of methods for using retroviral vectors forgene therapy are described in U.S. Pat. Nos. 4,868,116 and 4,980,286;PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science260:926-932 (1993)); the teachings of which are incorporated herein byreference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology51:261-21 A (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the Elgene removed and these virions are generated in a cell line such as thehuman 293 cell line. In another embodiment both the El and E3 genes areremoved from the adenovirus genome.

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B 19 coding regions have been deleted, resultingin a safe, non-cytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

The disclosed vectors thus provide DNA molecules which are capable ofintegration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral vectors usually containpromoters, and/or enhancers to help control the expression of thedesired gene product. A promoter is generally a sequence or sequences ofDNA that function when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Molecular genetic experiments with large human herpes viruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature Genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable the maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpes virus amplicon systems arealso being used to package pieces of DNA>220 kb and to infect cells thatcan stably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating Vaccinia virus vectors.

In embodiments, compositions disclosed herein can be delivered to targetcells in a variety of ways. For example, the compositions can bedelivered through electroporation, or through lipofection, or throughcalcium phosphate precipitation. The delivery mechanism chosen willdepend in part on the type of cell targeted and whether the delivery isoccurring for example in vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosed FS1-78, FS 6-21, FS SMC1A, and other disclosed novopeptides, vectors, suchas, for example, lipids such as liposomes, which may include cationicliposomes (e.g., DOTMA, DOPE, DC-cholesterol) and/or anionic liposomes.Liposomes can further comprise proteins to facilitate targeting aparticular cell, if desired. Administration of a composition comprisinga compound and a cationic liposome can be administered to the bloodafferent to a target organ or inhaled into the respiratory tract totarget cells of the respiratory tract. Regarding liposomes, see, e.g.,Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner etal. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No.4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Lie. (San Diego,Calif.) as well as by means of a SONOPORATION machine (hnaRxPharmaceutical Corp., Tucson, Ariz.).

In embodiments, the materials may be in solution or suspension (forexample, incorporated into microparticles, liposomes, or cells). Thesemay be targeted to a particular cell type via antibodies, receptors, orreceptor ligands. The following references are examples of the use ofthis technology to target specific proteins to tumor tissue (Senter, etal., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J.Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703,(1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, etal., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz andMcKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al.,Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be usedfor a variety of other specific cell types. Vehicles such as “stealth”and other antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis have been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome typically contain integration sequences. Thesesequences are often viral related sequences, particularly when viralbased systems are used. These viral integration systems can also beincorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of delivery, such as a liposome, so thatthe nucleic acid contained in the delivery system can become integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

In embodiments, compositions disclosed herein can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

Promoters controlling transcription from vectors in mammalian host cellsmay be obtained from various sources, for example, the genomes ofviruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HmdlH E restriction fragment (Greenway, P J. et al., Gene 18: 355-360(1982)). Of course, promoters from the host cell or related species alsoare useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. ScL 78:993 (1981)) or 3′ (Lusky,M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit.Furthermore, enhancers can be within an intron (Banerji, J. L. et al.,Cell 33: 729 (1983)) as well as within the coding sequence itself(Osborne, T. F., et al., Mol. Cell Bio. 4: 1293 (1984)). They areusually between 10 and 300 bp in length, and they function in cis.Enhancers function to increase transcription from nearby promoters.Enhancers also often contain response elements that mediate theregulation of transcription. Promoters can also contain responseelements that mediate the regulation of transcription. Enhancers oftendetermine the regulation of expression of a gene. While many enhancersequences are now known from mammalian genes (globin, elastase, albumin,-fetoprotein and insulin), typically one will use an enhancer from aeukaryotic cell virus for general expression. Preferred examples are theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

The promoter and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed, in certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTF.

It has been shown that regulatory elements can be cloned and used toconstruct expression vectors that are selectively expressed in specificcell types such as melanoma cells. The glial fibrillary acetic protein(GFAP) promoter has been used to selectively express genes in cells ofglial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs, in certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

In embodiments, viral vectors may include nucleic acid sequence encodinga marker product. This marker product is used to determine if the genehas been delivered to the cell and once delivered is being expressed.Example marker genes are the E. Coli lacZ gene, which encodesP-galactosidase, and green fluorescent protein.

In some embodiments a marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

A second category is dominant selection, which refers to a selectionscheme that may be used in any cell type and does not require the use ofa mutant cell line. These schemes typically use a drug to arrest growthof a host cell. Those cells which have a novel gene would express aprotein conveying drug resistance and would survive the selection.Examples of such dominant selection use the drugs neomycin, (Southern P.and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid,(Mulligan, R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin,(Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). These threeexamples employ bacterial genes under eukaryotic control to conveyresistance to the appropriate drug G418 or neomycin (geneticin), xgpt(mycophenolic acid) or hygromycin, respectively. Others include theneomycin analog G418 and puramycin.

In embodiments, compositions disclosed herein may be administered invivo in a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to a subject, alongwith the nucleic acid or vector, without causing unacceptable biologicaleffects or interacting in a deleterious manner with other components ofthe pharmaceutical composition in which it is contained. The carrier maybe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject, as would be well knownto one of skill in the art.

In embodiments, compositions disclosed herein may be administeredorally, parenterally (e.g., intravenously), by intramuscular injection,by intraperitoneal injection, transdermally, extracorporeally, topicallyor the like, including topical intranasal administration oradministration by inhalant. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. Administration of thecompositions by inhalant can be through the nose or mouth via deliveryby a spraying or droplet mechanism. Delivery can also be directly to anyarea of the respiratory system (e.g., lungs) via intubation. The exactamount of the compositions required will vary from subject to subject,depending on the species, age, weight and general condition of thesubject, the severity of the allergic disorder being treated, theparticular nucleic acid or vector used, its mode of administration andthe like. Thus, it is not possible to specify an exact amount for everycomposition. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

In embodiments, materials to be administered may be in solution orsuspension (for example, incorporated into microparticles, liposomes, orcells). These may be targeted to a particular cell type via antibodies,receptors, or receptor ligands. The following references are examples ofthe use of this technology to target specific proteins to tumor tissue(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D.,Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer,58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993);Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992);Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); andRoffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehiclessuch as “stealth” and other antibody conjugated liposomes (includinglipid mediated drug targeting to colonic carcinoma), receptor mediatedtargeting of DNA through cell specific ligands, lymphocyte directedtumor targeting, and highly specific therapeutic retroviral targeting ofmurine glioma cells in vivo. The following references are examples ofthe use of this technology to target specific proteins to tumor tissue(Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger andHuang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis have been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

In embodiments, compositions disclosed herein, including antibodies, canbe used therapeutically in combination with a pharmaceuticallyacceptable carrier. Suitable carriers and their formulations aredescribed in Remington: The Science and Practice of Pharmacy (19th ed.)ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically,an appropriate amount of a pharmaceutically-acceptable salt is used inthe formulation to render the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

In embodiments, compositions disclosed herein may be administered in orwith pharmaceutical compositions, which may include carriers,thickeners, diluents, buffers, preservatives, surface active agents andthe like in addition to the molecule of choice. Pharmaceuticalcompositions may also include one or more active ingredients such asantimicrobial agents, anti-inflammatory agents, anesthetics, and thelike.

In embodiments, pharmaceutical compositions may be administered in anumber of ways depending on whether local or systemic treatment isdesired, and on the area to be treated. Administration may be topically(including ophthalmically, vaginally, rectally, intranasally), orally,by inhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

In embodiments, compositions may be administered as or with apharmaceutically acceptable acid-or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

In embodiments, effective dosages and schedules for administeringcompositions disclosed herein may be determined empirically, and makingsuch determinations is within the skill in the art. In embodiments, adosage range for the administration of the compositions may include adosage large enough to produce a desired effect in which thesymptoms/disorder are/is affected. In embodiments, a dosage shouldpreferably not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like, althoughadverse effects cannot always be avoided and may be outweighed by thebenefits sought. In embodiments, dosage may vary with the age,condition, sex and extent of the disease in the patient, route ofadministration, or whether other drugs are included in the regimen, andcan be determined by one of skill in the art. The dosage can be adjustedby the individual physician in the event of any counterindications.Dosage can vary, and can be administered in one or more doseadministrations daily, or according to any dosing schedule foundbeneficial. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. For example,guidance in selecting appropriate doses for antibodies can be found inthe literature on therapeutic uses of antibodies, e.g., Handbook ofMonoclonal Antibodies, Ferrone et al., eds., Noges Publications, ParkRidge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York(1977) pp. 365-389. A typical daily dosage of an antibody used alonemight range from about 1 mg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

Following administration of a disclosed composition, such as a vaccineor an antibody, for treating, inhibiting, or preventing a cancer, theefficacy of the therapy or prophylaxis can be assessed in various wayswell known to the skilled practitioner. For instance, one of ordinaryskill in the art will understand that a composition, such as a vaccineor an antibody, disclosed herein is efficacious in treating, inhibiting,or preventing a cancer in a subject by observing that the compositionreduces tumor growth or prevents a further increase in tumor size.

Also disclosed herein are embodiments of kits that are drawn to reagentsthat can be used in practicing the methods disclosed herein. The kitscan include any reagent or combination of reagent discussed herein orthat would be understood to be required or beneficial in the practice ofthe disclosed methods. For example, the kits could include primers toperform the amplification reactions discussed in certain embodiments ofthe methods, as well as the buffers and enzymes required to use theprimers as intended. For example, disclosed is a kit for assessing asubject's risk for acquiring prostate cancer, comprising the peptidesset forth in SEQ ID Nos: 2, 4, and 8.

In embodiments, there is provided an anti-cancer vaccine, to beadministered prophylactically or therapeutically or both to a populationcomprising a plurality of mammalian subjects in accordance with thedisclosure hereof, useful for reducing the susceptibility of thepopulation to at least one cancer. (The use of the word ‘vaccine’ is notintended to limit the disclosure hereof to a particular mechanism ofaction; although there is sound theoretical and experimental support foran immunological basis for their effectiveness, the disclosure hereofextends to the disclosed compositions and methods regardless of theirmechanism of action.) Reducing the susceptibility of the population toat least one cancer may include any improvement in the health status ofat least one subject relative to at least one cancer, such as, by way ofexample only, a temporary or permanent reduction in the likelihood of atleast one member of the population contracting a cancer; a temporary orpermanent reduction in the incidence rate of a cancer in the population;a temporary or permanent improvement in the average severity,amenability to treatment, and/or average prognosis of the cancer in thepopulation; a temporary or permanent reduction in a rate of tumorprogression in at least one member of the population; an improvement inthe efficacy of a cancer treatment in at least one member of thepopulation; and/or an improvement in a survival rate in the populationrelative to at least one cancer; and/or an improvement in the survivaltime of at least one member of the population after contracting acancer.

In some embodiments, an anti-cancer vaccine may include at least onepeptide component. In embodiments, a peptide component of an anti-cancervaccine may include a continuous synthetic or recombinant amino acidchain, from about 8 to about 40 amino acids in length, having at least75%, or 80%, or 85%, or 90%, or 95%, or 99% sequence identity over itslength with a continuous amino acid sequence (or continuous portionthereof) according to the formula D1-D2, wherein D1 and D2 are aminoacid sequences each encoded by a different nucleic acid sequence orportion thereof of a genome of a mammalian species for which the vaccineis intended, wherein D1 is read in its wild-type reading frame, and D2is read in a non-wild-type reading frame. In some embodiments, thenucleic acid sequence encoding D2 is not contiguous, in the genome ofthe mammalian species, with the nucleic acid sequence encoding D1; forexample, the nucleic acid sequence encoding D2 may comprise all or partof a different gene, a different exon, a non-coding region, a differentnucleic acid strand, and/or a different chromosome, relative to thenucleic acid sequence encoding D1. A synthetic or recombinant amino acidchain may include and extend to any non-naturally occurring amino acidchain, such as, for example, an amino acid chain produced by chemicalsynthesis, by an expression system, by in vitro translation, bychemical, physical, or other isolation, by inducing expression in a hostcell such as by genetic immunization, or by any other method resultingin an amino chain having a sequence and/or other characteristics notobtainable from nature.

It will be apparent to persons of skill in the art that the functionalcharacteristics of a peptide may typically be retained in whole or partby other peptides having similar or related sequences, such as whereinsertions, deletions, and/or substitutions are made that are not sosignificant in character or degree to disrupt the interactions givingrise to the function of interest. The disclosure hereof extends toanti-cancer vaccine peptide components comprising peptide sequenceswithin the range of similarity to the disclosed sequences that would beregarded by persons of skill in the art as of probable full or partialfunctional similarity to any of the disclosed sequences. In someembodiments, an anti-cancer vaccine may include in lieu of a peptidecomponent as disclosed herein, a biochemically acceptable substitute. Abiochemically acceptable substitute may include any molecular entitythat a person of skill in the art would regard as functionally similaror equivalent to a peptide component disclosed herein, such as, forexample, a peptide having, relative to a peptide component disclosedherein, an alignment score, obtained according to an art-recognizedsequence alignment method, that a person of skill in the art wouldregard as implying likely full or partial functional similarity, or apeptide differing from a peptide component disclosed herein by reason offewer than 10, or fewer than 7, or fewer than 5, or fewer than 4, orfewer than 3, or fewer than 2, or fewer than 1 conservativesubstitutions, or any of the other molecular entities disclosedelsewhere herein or recognized in the art as fully or partiallyfunctionally equivalent in their biochemical characteristics to apeptide component disclosed herein.

In embodiments, there is provided a method of reducing thesusceptibility of a population including a plurality of mammaliansubjects to at least one cancer, the method including administering toat least one member of the population an effective dose of ananti-cancer vaccine.

In some embodiments an anti-cancer vaccine may be administered by anymethod of administration operable to expose the organism to bevaccinated to an effective dose of the vaccine, such as, for example, bysubcutaneous, intramuscular, or other direct administration of anembodiment of the anti-cancer vaccine including one or more peptidecomponents in a pharmaceutically acceptable carrier, and optionallyaccompanied by a suitable adjuvant, according to an effectiveadministration protocol. In some embodiments, an anti-cancer compositionmay be administered as a genetic vaccine, and/or may include, forexample, a nucleic acid sequence encoding one or more peptidecomponents. In some embodiments, the nucleic acid sequence may form partof a vector, plasmid, LEE, or other entity whereby a cell of themammalian subject may be induced to express the peptide component. Thedisclosure hereof extends to nucleic acid sequences adapted foradministration by genetic immunization and/or for inclusion in a vector,plasmid, LEE, or other entity or carrier adapted for geneticimmunization or for expression in an expression system, encoding any ofthe peptide components disclosed herein, and to vectors, plasmids,LEE's, or other entities comprising the nucleic acid sequences. By wayof example, the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQID NO:239, and SEQ ID NO:241 are disclosed as nucleic acid sequencesthat in whole or part encode the peptide component sequences SEQ IDNO:294, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:232, SEQ ID NO:234, SEQ IDNO:236, SEQ ID NO:238, SEQ ID NO:240, and SEQ ID NO:242, respectively.

An effective dose of an anti-cancer vaccine may be any quantity operableto reduce the susceptibility of a mammalian subject to which ananti-cancer vaccine is administered, or a population to which thesubject belongs, to at least one cancer. In some embodiments, aneffective dose of an anti-cancer vaccine may include any quantityoperable to expose the immune system of a subject to a peptide componentthereof. By way of example only, an effective dose of an anti-cancervaccine administered in the form of synthetic or recombinant peptides ina carrier may be a quantity operable to produce in the subject anantibody response, which may be detected, for example, by an ELISA orother assay confirming the presence in serum of the subject antibodiesspecific for one or more of the peptide components. In some embodiments,for example, an effective dose of an anti-cancer vaccine foradministration in the form of peptides in or on a carrier may be atleast 50 μg KLH-conjugated peptide, or at least 100 μg KLH-conjugatedpeptide, or at least 200 μg KLH-conjugated peptide. By way of exampleonly, an effective dose of an anti-cancer vaccine administered in theform of a genetic vaccine including nucleic acid(s) encoding the peptidecomponents may be a quantity operable to cause expression of at leastone peptide component in a cell, such as, for example, an antigenpresenting cell, of the subject, and display of the peptide component ora fragment thereof in MHC. In embodiments, for example, an effectivedose for genetic immunization by gene gun may be at least 20 ng perbullet, or at least 50 ng per bullet, or at least 100 ng per bullet, inat least one shot, or at least two shots, or at least four shots, or atleast six shots.

In some embodiments of an anti-cancer vaccine, it may be found useful toselect peptide components having a cancer association ratio of at least2:1, or at least 3:1, or at least 4:1, or at least 5:1. A cancerassociation ratio of a peptide component may be employed as a measure ofthe presence of the peptide component (or its homolog or equivalent) incancer tissue relative to its presence in comparable non-canceroustissue. Thus a peptide that presents an epitope abundantly present inidentical or recognizable form in cancerous tissue and rarely or neverpresent in comparable non-cancerous tissue would have a relatively highcancer association ratio, making it a desirable candidate as a peptidecomponent of a vaccine. In embodiments, the cancer association ratio ofa peptide component may be measured or estimated in any way operable toyield an informative indication, of the degree of presence of an epitopeequivalent or immunologically similar to an epitope of the peptidecomponent in a cancerous tissue of interest, relative to a comparablenon-cancerous tissue. In some embodiments, a cancer association ratiomay preferably be determined and/or computed in a manner that would beregarded by a person of skill in the art as providing acceptablestatistical and experimental validity. In some embodiments, for example,a cancer association ratio may be estimated by measuring or estimating alevel of expression of the sequence (or relevant portion or homologthereof) in mRNA transcripts in cells of a tissue sample of the cancertype of interest, making a similar measurement or estimate in cells of asample of non-cancerous tissue of similar or comparable type, and takingthe ratio of the two. In some embodiments, a cancer association ratiomay be measured or estimated as an odds ratio, computed as disclosed inExample 5 herein. In some embodiments, a cancer association ratio may bemeasured or estimated by determining the prevalence of the sequence (orrelevant portion or homolog thereof) in a cancer EST library ordatabase, as compared to a non-cancer EST library or database. In someembodiments wherein it would be apparent from art-accepted principles ofmolecular biology that the sequence of interest would not be expressedby normal cells, a cancer association ratio may be assumed to be >>1 ifsignificant presence of the sequence is confirmed in the canceroustissue of interest, or in mRNA or cDNA thereof.

In some embodiments, of an anti-cancer vaccine, it may be found usefulto select peptide components having a cancer serum recognitionpercentage of at least 40%, or at least 60%, or at least 80%. A cancerserum recognition percentage may be determined or estimated in any wayoperable to provide an informative and reasonably repeatable measure, ina mammalian population cohort consisting of subjects having a cancer, ofthe percentage of subjects whose serum shows specific antibodyreactivity to the peptide component or a portion or homolog thereof. Inembodiments, such reactivity may be measured or estimated in anyoperable way, such as, for example, by ELISA or by microarray assay. Inembodiments, the population cohort should preferably include arepresentative selection and quantity of subjects, such as, for example,at least 10, or at least 20, or at least 30 independent samples fromdistinct cancerous individuals. In embodiments, a cancer serumrecognition percentage should preferably be determined and/or computedin a manner that would be regarded by a person of skill in the art asproviding acceptable statistical and experimental validity.

In some embodiments, there is provided a peptide component of ananti-cancer vaccine to be administered to a mammalian subject belongingto a first mammalian species, having a sequence determined by homologyto a peptide component whose utility has been assessed relative to asecond mammalian species recognized in the art as an appropriate modelwith respect to the first mammalian species. Thus, by way of exampleonly, a peptide component for use in a human vaccine may have a sequencedetermined in whole or part by reference to a peptide component that hasbeen assessed in a mouse model, by combining the non-frame-shifted humansequence corresponding to the homologous gene or gene fragmentcomprising the non-frame-shifted portion of the mouse peptide componentwith the frame-shifted human sequence corresponding to the nucleic acidsequence homologous to the gene or gene fragment from which theframe-shifted portion of the mouse peptide component is transcribed.Accordingly, where peptide components are disclosed herein correspondingto novopeptides relating to human, mouse, or dog sequences, thedisclosure hereof extends to the homologous counterparts of thesepeptide components in the others of these species. Thus, for example, inaddition to the mouse-targeted sequences FS 1-78 (SEQ ID NO: 294), FS6-21 (SEQ ID NO: 291), FS SMC1A (SEQ ID NO: 297), FS RBM (SEQ ID NO:232), and FS THAP2 (SEQ ID NO: 238) as disclosed, also disclosed hereinare the corresponding sequences for human (SEQ ID NO: 293, SEQ ID NO:290, SEQ ID NO: 296, SEQ ID NO: 234, and SEQ ID NO: 240, respectively)and dog (SEQ ID NO: 295, SEQ ID NO: 292, SEQ ID NO: 298, SEQ ID NO: 236,and SEQ ID NO: 242, respectively).

In some embodiments there is provided an anti-cancer vaccine including apeptide component or a plurality of distinct peptide components, and apharmaceutically acceptable carrier. Peptide components may includepeptide chains as disclosed herein which may be in any form operable foradministration as disclosed herein, such as, for example, free and/orunbound peptide chains in a carrier solution or suspension, peptidechains bound or associated to other entities, or peptide chainsconjugated to other entities, such as KLH-conjugated peptides. Carriersmay be any pharmaceutically acceptable entities or compositions operableto contain, suspend, bind to, or otherwise associate with peptidecomponents for administration to a mammalian subject. In someembodiments there is provided an anti-cancer vaccine for administrationby genetic immunization, including a nucleic acid or a plurality ofnucleic acids, encoding a peptide component or a plurality of peptidecomponents, and a carrier adapted for administration of the anti-cancervaccine by genetic immunization, which may include any pharmaceuticallyacceptable entity or composition operable to facilitate theadministration of the nucleic acid(s) to a mammalian subject whereby thepeptide components encoded by the nucleic acids may be expressed bycells of the subject. Examples include vectors, plasmids, LEE's, orother entities incorporating sequences or functionality for facilitatingsuch expression, which may in some embodiments be applied, adsorbed,conjugated to, or otherwise associated with a physical delivery entitysuch as a gold nanoparticle or other genetic immunization ‘bullet’.

In some embodiments an anti-cancer vaccine includes a plurality ofpeptide components, or a nucleic acid portion encoding peptidecomponents, wherein each peptide component is targeted to a differentcancer type, so that the susceptibility of a receiving subject orpopulation may be reduced with respect to more than one cancer type. Insome embodiments an anti-cancer vaccine includes a plurality of peptidecomponents, or a plurality of nucleic acids encoding peptide components,wherein the peptide components are displayable in different MHC or HLAtypes, so that the coverage of the anti-cancer vaccine extends tosubjects of more than one MHC or HLA type. In some embodiments, it maybe found useful to confirm by art-recognized computational methods thata peptide component is displayable in MHC of a type expressed by amammalian subject, which may be a member of a population for which avaccine composition is intended. In embodiments, a population for whicha vaccine composition is intended may be the population of all mammalsof a species or subspecies of interest, or may be a sub-populationdefined by one or more characteristics of interest such as, for example,a sub-population having a particular genetic trait, or a particularsusceptibility. In some embodiments, a peptide component may bedisplayable by MHC of a type expressed by a minimum percentage of apopulation of a mammalian species or sub-population for which ananti-cancer vaccine is intended, such as, for example, at least 2%, orat least 4%, or at least 6%, or at least 10%, or at least 20% of thepopulation or sub-population. In some embodiments, an anti-cancervaccine may include two or more peptide components (or nucleic acid(s)encoding them, in the case of a genetic vaccine), wherein a firstpeptide components is displayable by a first MHC type expressed in onecohort of the target population or sub-population, and a second peptidecomponent is displayable by a second MHC type expressed in a differentcohort of the target population or sub-population, so that the potentialcoverage of the vaccine extends to both cohorts. It will be apparentthat by appropriate selection of peptide components displayable bydifferent MHC types, it is possible for a cocktail vaccine to extend thebenefit of the vaccine to an arbitrary proportion of the targetpopulation.

In some embodiments, disclosed herein is a synthetic or recombinantpeptide component for an anti-cancer vaccine for administration in apopulation of a mammalian species, including a continuous amino acidchain, or a continuous portion thereof from 8 to 40 amino acids inlength, according to the formula D1-D2, wherein D1 and D2 each have atleast threshold identity to a polypeptide sequence encoded by adifferent exon or continuous portion thereof of the genome of themammalian species, wherein D1 is encoded in a wild type reading frameand D2 is encoded in a non-wild type reading frame. In embodiments, athreshold identity may be any level of sequence identity or similarity,determined according to metric thereof, that would be regarded by aperson of skill in the art as providing a reasonable measure offunctional similarity, such as, by way of example only, 75% sequenceidentity, or 80% sequence identity, or 85% sequence identity, or 90%sequence identity, or 95% sequence identity.

In some embodiments of a peptide component, the portion of D1immediately adjacent to D2 is not a microsatellite or portion thereof.In some embodiments neither D1 nor D2 has above a threshold identity toa peptide sequence encoded by an oncogene. In some embodiments of apeptide component, the RefSeq of the mammalian species does not containa nucleic acid sequence encoding the sequence of the peptide component.In some embodiments of a peptide component, the normal transcriptome ofthe mammalian species does not contain a nucleic acid sequence encodingthe sequence of the peptide component. In some embodiments of a peptidecomponent, the peptide component aligns with at least threshold identityto all or a portion of an mRNA transcript expressed in at least onecancer type of the mammalian species. In some embodiments of a peptidecomponent, the peptide component has a cancer association ratio of atleast 2:1 with respect to at least one cancer type of the mammalianspecies. In some embodiments of a peptide component, the peptidecomponent has a cancer serum recognition percentage of at least 40% withrespect to at least one cancer type of the mammalian species. In someembodiments of a peptide component, the peptide component is capable ofbeing displayed in a Class I major histocompatibility complex (MHC-I) ofa type expressed by at least 4 percent of the population.

In some embodiments of a peptide component, the mammalian species ismouse and the continuous amino acid chain has at least thresholdsequence identity to a sequence selected from: peptide FS 1-78 (SEQ IDNO: 294), peptide FS 6-21 (SEQ ID NO:291), peptide FS SMC1A (SEQ ID NO:297), peptide FS RBM (SEQ ID NO: 232), or peptide FS THAP2 (SEQ ID NO:238). In some embodiments of a peptide component, the mammalian speciesis human and the continuous amino acid chain has at least thresholdsequence identity to a sequence selected from: peptide FS 1-78 (SEQ IDNO: 293), peptide FS 6-21 (SEQ ID NO: 290), peptide FS SMC1A (SEQ ID NO:296), peptide FS RBM (SEQ ID NO: 234), or peptide FS THAP2 (SEQ ID NO:240). In some embodiments of a peptide component, the mammalian speciesis dog and the continuous amino acid chain has at least thresholdsequence identity to a sequence selected from: peptide FS 1-78 (SEQ IDNO: 295), peptide FS 6-21 (SEQ ID NO: 292), peptide FS SMC1A (SEQ ID NO:298), peptide FS RBM (SEQ ID NO: 236), or peptide FS THAP2 (SEQ ID NO:242).

Also disclosed herein are embodiments of an anti-cancer vaccine foradministration in a population of a mammalian species, including aplurality of any of the peptide components disclosed herein, in apharmaceutically acceptable carrier; also disclosed herein areembodiments of a method including administering to a mammal an effectivedose of the anti-cancer vaccine. Also disclosed herein are embodimentsof an anti-cancer vaccine for administration in a population of amammalian species, including a synthetic or recombinant nucleic acid orplurality thereof encoding a plurality of any of the peptide componentsdisclosed herein, and a carrier adapted for administration of theanti-cancer vaccine by genetic immunization; also disclosed herein areembodiments of a method including administering to a mammal an effectivedose of the anti-cancer vaccine, which may employ genetic immunizationmethods. In some embodiments of an anti-cancer vaccine, there isprovided a plurality of peptide components including a first peptidecomponent displayable in a first MHC type expressed in a first cohort ofthe population and a second peptide component displayable in a secondMHC type expressed in a second cohort of the population, wherein thefirst and second cohort together are more numerous by at least 2 percentof the population than either cohort.

In some embodiments, there is disclosed a method including:administering to a mammal by genetic immunization an effective dose ofan anti-cancer vaccine including a synthetic or recombinant nucleic acidor plurality thereof, encoding a plurality of any of the peptidecomponents as disclosed herein, and a carrier adapted for administrationof the anti-cancer vaccine by genetic immunization; and at least twoweeks thereafter, administering to the mammal an effective dose of ananti-cancer vaccine including at least one of the plurality of peptidecomponents in a pharmaceutically acceptable carrier.

In embodiments, disclosed herein is a composition including synthetic orrecombinant peptides each including a continuous amino acid chain, orcontinuous portion thereof from 8 to 40 amino acids in length, having atleast threshold identity to a sequence or continuous portion thereof ofSEQ ID NO: 310, SEQ ID NO: 311, and SEQ ID NO: 312. Also disclosed areembodiments of a synthetic or recombinant nucleic acid or pluralitythereof encoding peptides each including a continuous amino acid chain,or continuous portion thereof from 8 to 40 amino acids in length, havingat least threshold identity to a sequence or continuous portion thereofof SEQ ID NO: 310, SEQ ID NO: 311, and SEQ ID NO: 312.

Also disclosed are embodiments of a synthetic or recombinant peptideincluding a continuous amino acid chain, or a continuous portion thereoffrom 8 to 40 amino acids in length, having at least threshold identityto a sequence or continuous portion thereof selected from the groupconsisting of SEQ ID NO: 240 and SEQ ID NO: 243 through SEQ ID NO: 289.Also disclosed are embodiments of a synthetic or recombinant nucleicacid encoding a continuous amino acid chain, or a continuous portionthereof from 8 to 40 amino acids in length, having at least thresholdidentity to a sequence or continuous portion thereof selected from thegroup consisting of SEQ ID NO: 240 and SEQ ID NO: 243 through SEQ ID NO:289. Also disclosed are embodiments of a synthetic or recombinantpeptide including a continuous amino acid chain, or a continuous portionthereof from 8 to 40 amino acids in length, having at least thresholdidentity to a sequence or continuous portion thereof selected from thegroup consisting of SEQ ID NO: 242, SEQ ID NO: 236, and SEQ ID NO: 306through SEQ ID NO: 309. Also disclosed are embodiments of a synthetic orrecombinant nucleic acid encoding a continuous amino acid chain, or acontinuous portion thereof from 8 to 40 amino acids in length, having atleast threshold identity to a sequence or continuous portion thereofselected from the group consisting of SEQ ID NO: 242, SEQ ID NO: 236,and SEQ ID NO: 306 through SEQ ID NO: 309. Also disclosed areembodiments of a synthetic or recombinant peptide including a continuousamino acid chain, or a continuous portion thereof from 8 to 40 aminoacids in length, having at least threshold identity to a sequence orcontinuous portion thereof selected from the group consisting of SEQ IDNO: 238, SEQ ID NO: 242, and SEQ ID NO: 299 through SEQ ID NO: 305. Alsodisclosed are embodiments of a synthetic or recombinant nucleic acidencoding a continuous amino acid chain, or a continuous portion thereoffrom 8 to 40 amino acids in length, having at least threshold identityto a sequence or continuous portion thereof selected from the groupconsisting of SEQ ID NO: 238, SEQ ID NO: 242, and SEQ ID NO: 299 throughSEQ ID NO: 305. Also disclosed are embodiments of a compositionincluding synthetic or recombinant peptides each including a continuousamino acid chain, or continuous portion thereof from 8 to 40 amino acidsin length, having at least threshold identity to a sequence orcontinuous portion thereof of SEQ ID NO: 310, SEQ ID NO: 311, and SEQ IDNO: 312. Also disclosed are embodiments of a synthetic or recombinantnucleic acid or plurality thereof encoding peptides each including acontinuous amino acid chain, or continuous portion thereof from 8 to 40amino acids in length, having at least threshold identity to a sequenceor continuous portion thereof of SEQ ID NO: 310, SEQ ID NO: 311, and SEQID NO: 312. Also disclosed are embodiments of a synthetic or recombinantpeptide including a continuous amino acid chain, or a continuous portionthereof from 8 to 40 amino acids in length, having at least thresholdidentity to a sequence or continuous portion thereof selected from thegroup consisting of SEQ ID NO: 310 through SEQ ID NO: 332. Alsodisclosed are embodiments of a synthetic or recombinant nucleic acidencoding a continuous amino acid chain, or a continuous portion thereoffrom 8 to 40 amino acids in length, having at least threshold identityto a sequence or continuous portion thereof selected from the groupconsisting of SEQ ID NO: 310 through SEQ ID NO: 332. Also disclosed areembodiments of a synthetic or recombinant peptide including a continuousamino acid chain, or a continuous portion thereof from 8 to 40 aminoacids in length, having at least threshold identity to a sequence orcontinuous portion thereof selected from the group consisting of SEQ IDNO: 333 through SEQ ID NO: 341. Also disclosed are embodiments of asynthetic or recombinant nucleic acid encoding a continuous amino acidchain, or a continuous portion thereof from 8 to 40 amino acids inlength, having at least threshold identity to a sequence or continuousportion thereof selected from the group consisting of SEQ ID NO: 333through SEQ ID NO: 341. Also disclosed are embodiments of a synthetic orrecombinant peptide including a continuous amino acid chain, or acontinuous portion thereof from 8 to 40 amino acids in length, having atleast threshold identity to a sequence or continuous portion thereofselected from the group consisting of and SEQ ID NO: 342 through SEQ IDNO: 348. Also disclosed are embodiments of a synthetic or recombinantnucleic acid encoding a continuous amino acid chain, or a continuousportion thereof from 8 to 40 amino acids in length, having at leastthreshold identity to a sequence or continuous portion thereof selectedfrom the group consisting of SEQ ID NO: 342 through SEQ ID NO: 348.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in degrees Celsiusor is at ambient temperature, and pressure is at or near atmospheric.

Example 1

To determine the amount of coding sequence that must be sequenced toidentify a sufficient number of novopeptides that are expressed in tumorcells and not in non-cancerous cells to produce a practicable cancervaccine, it is first instructive to determine the frequency ofnovopeptide associated mutation or variation in a tumor. To assess thisfrequency, the C-terminal 600 bp of 550 genes that are expressed in themouse melanoma cell line, B16-F10 were sequenced. To confirm that the invitro-derived FS were expressed in vivo, RNA was extracted from B16 lungmetastases after injection of cells systemically, cDNA was generated,and the FS confirmed by RT-PCR sequencing. As illustrated in FIG. 2 a,cDNA was generated from RNA isolated from cultured cells. It wassequenced directly by RT-PCR sequencing. FIG. 2b illustrates in vivoconfirmation of in vitro-derived FS mutations. To confirm that the FSwas expressed in vivo, RNA was extracted from B16 lung metastases afterinjection of cells systemically. cDNA was generated and the FS confirmedby RT-PCR sequencing.

Three novopeptides were isolated, indicating that FS occur at afrequency of roughly one per 183 segments of 600 bp of genes (FIG. 2).FS 1-78 (SEQ ID NO: 2) was identified as a frameshift relative to thenormal reference sequence. One other frameshift peptide, FS 6-21 (SEQ IDNO: 4) was also identified, as was a 3 amino acid insertion. The parentprotein of FS 1-78 is a zinc finger protein, but a deletion of 396 bpresults in expression of an 11 amino acid novopeptide in an alternateframe before termination. Table 1 shows the sequences identified.Underlined amino acids comprise the peptide predicted to be presented byH-2 Db (C57BL6 mice) and H-2 Kd (Balb/c mice). Upper case amino acidsindicate primary frame and lower case amino acids indicate the frameshift residues. Fusions of primary and alternate frames often formantigenic peptides. FIG. 1a shows PCR amplification of FS 1-78. Arrowindicates FS 1-78 band; other bands are wild type alleles. Lane 1:B16/F10 tumor cells; Lane 2: B16/F10 tumor cells; Lane 3: normal heart;Lane 4: normal intestine; Lane 5: normal kidney; Lane 6: normal liver;Lane 7: normal lung; Lane 8: normal skeletal muscle; Lane 9: normalskin; Lane 10: normal spleen; Lane M: Molecular weight marker. FIG. 1bshows the analysis of the occurrence of the FS 6-21 frameshift in themouse tumor versus RNA from normal tissue. Arrow indicates FS 6-21 FSband. Lanes are as in FIG. 1 a. It is noted that FS 1-78 expression isdetected in the tumor cells and not in any of the noncancerous cellstested.

TABLE 1 Gene FS SEQ ID Name mutation Novopeptide Sequence NO 1-78396 bp  IPRMQPQASAnhcqllkvmva* SEQ ID  deletion NO: 2 6-21  95 bp AVLLMCQLYQpwmckeyyrll* SEQ ID  deletion NO: 4 3-83   3 aa . . . GTEDsrdSDDALL . . . SEQ ID  insertion NO: 6

Example 2

The FS 1-78 novopeptide identified above was chemically synthesized as agenetic linear expression element (LEE) as diagrammed in FIG. 3according to the methods described in (Sykes, K. F., and S. A. Johnston(1999) Nat. Biotechnol. 17:355). Each LEE comprises a fragment that maycontain a mammalian promoter 301, a ubiquitin gene (Ub) 303 for strongerintracellular processing, and a fragment 305 that containstranscriptional and translational terminators. The two fragments arelinked via the frameshift sequence 307, here FS 1-78. Using gene guntechnology, C57BL6 mice were then genetically immunized with the FS 1-78LEE construct in doses as shown in FIG. 4 and a plasmid expressingGM-CSF (1 μg of pGM-CSF). Mice were boosted 2 weeks later with the sameFS 1-78 LEE and pGM-CSF and then challenged one week after boost (day 0)with 1×10⁵ B16 F10 melanoma tumor cells. As shown in FIG. 4, tumorgrowth was markedly delayed compared to mice administered control emptyLEE and compared to those receiving no immunization, and at the highestdose (3.2 ng), tumor volume decreased after the already delayed rise.

Example 3

Immunization with a single FS-novopeptide identified based on one tumortype may be immunoprotective against a different tumor type in adifferent mouse strain. Disclosed herein is a procedure for suchimmunization. Immunization with novopeptides common to multiple types oftumors can result in cross-protection, obviating the requirement that apatient must develop a tumor before a personalized vaccine can beformulated, prepared and administered. Balb/c mice were immunized in thesame manner as the C57BL6 mice in Example 2 above, with 3.2 ng of FS1-78 LEE+1 μg of pGM-CSF, and boosted with the same gene vaccine after 2weeks. One week after the boost, mice were challenged with 1×10⁴ 4T1breast tumor cells. Seventeen days after 4T1 challenge, tumors startedto grow. As shown in FIG. 5, prophylactic immunization of Balb/c micewith FS 1-78 novopeptide significantly delayed and reduced 4T1 tumorgrowth in comparison to both controls immunized with pGM-CSF plasmidalone and controls immunized with another novopeptide (FS 6-21) that isnot found in 4T1 tumors.

Example 4

Vaccines combining more than one novopeptide can be highly effective inconferring immunoprotection against cancer. Mice were vaccinated using avaccine comprising a combination of FS 1-78 and FS 6-21 novopeptides. Onchallenge with B16 tumor cells, most vaccinated mice were completelyprotected from tumor growth. FIG. 6 compares the relative protection ofthe FS 1-78 and FS 6-21 peptides by themselves and when pooled as asingle vaccine. On Day −8, mice were immunized with the FS 6-21 peptide(squares), the FS 1-78 peptide (crosses), a combination of both(triangles), or an irrelevant peptide sequence (diamonds). Tumor cellswere implanted on Day 0. The 80 percent of mice in the group receivingthe combined vaccine that were alive on day 15 remained alive andapparently healthy thereafter and until the experiment was terminated.This experiment demonstrates that pooling of novopeptides can giveincreased protection over single peptide immunization.

Example 5

Candidate novopeptide nucleic acid sequences, expressed by cancerouscells and not by non-cancerous cells, can be identified and predicted bybioinformatics analysis comparing tumor database data with genomic data.This example illustrates an embodiment of the methods by which this wasdone. FS-novopeptide candidates were identified by bioinformaticanalysis of frame shifts by comparing sequences obtained from tumor andnormal EST library databases. Exact FS peptide sequences were thenconfirmed by DNA sequencing across the frame shift region and comparisonto the non-cancerous reference sequence. It is noteworthy that severalof the tumor-specific variants are not encoded at the DNA level butinvolve RNA splicing variants that are predominant in the tumors. Table2a shows frame shift sequences predicted by the bioinformatic comparisonand verified by DNA sequence analysis, and shows that these sequencesare present in the indicated number of tumor EST's and not innon-cancerous EST's.

TABLE 2a Gene FS EST Name mutation analysis Novopeptide Sequence RIPK2154 bp Tumor:  . . . HIHTPLLDrklnil SEQ ID deletion  6 of 16 mllgh*NO: 9 Normal:   0 of 8 DTYMK  91 bp Tumor:  . . . SANRWEQVifp* SEQ IDdeletion  3 of 86 NO: 10 Normal:   0 of 30 6-21  95 bp Tumor: . . . LLMCQCQLY  SEQ ID deletion NA Qpwmckeyyrll* NO: 11 Normal:  NADYRK4  61 bp Tumor:  . . . EQLACIMEipkvfl SEQ ID deletion  4 of 10 ki*NO: 12 Normal:   0 of 11 MTCH2  68 bp Tumor:  . . . SYSQAVTGscwwmpSEQ ID deletion  5 of 88 sllpniyvldrllvhatkrg NO: 13 Normal:  eyeprk* 0 of 63 FTH1  62 bp Tumor:  . . . ASYVYLSMivtatc SEQ ID insertion17 of 2157 lwgslv* NO: 14 Normal:   0 of 243

Bioinformatic identification of possible novopeptides was performed asfollows: the NCBI EST database was screened using information obtainedfrom the NCI EST database to classify each NCBI EST into one of threesets: tumor EST's, normal EST's and EST's for which there wasinsufficient information to classify as tumor or normal. The latter werediscarded. Each human reference sequence in the NCBI database was thenaligned with both the normal EST set and the tumor EST set using BLAST,and the number of frameshifted and unframeshifted hits of at least 100base pairs and 85% sequence identity were counted. To identifycandidates for further screening, an odds ratio was computed for each FSvariant sequence arising from an indel of at least 10 base pairs. Theodds ratio provides an indication of the relative expression of the FSvariant in tumor and normal cells, as compared to the expression of thenonvariant wild type sequence in tumor and normal cells. The ratio of FSvariants to wild type in tumor cells (the “tumor cell variant ratio”)was computed as the ratio of the number of sequence matches obtainedupon search of the tumor EST databases for the FS variant sequence tothe number of sequence matches obtained upon search of the tumor ESTdatabases for the wild type sequence. The ratio of FS variants to wildtype in normal cells (the “normal cell variant ratio”) was computed asthe ratio of the number of sequence matches obtained upon search of thenormal EST databases for the FS variant sequence to the number ofsequence matches obtained upon search of the normal EST databases forthe wild type sequence. In computing this ratio, the former number wasarbitrarily set to 1 if the number of matches were zero so as to avoiddivision by zero in computing the odds ratio; this approximation wasdeemed reasonable since the difference between zero and 1 is likelywithin the range of uncertainty associated with sequence alignment andthe setting of alignment parameters. An odds ratio was computed as theratio of the tumor cell variant ratio to the normal cell variant ratio,with FS variant sequences having a ratio above 2.0 being selected forfurther study. Table 2b shows six FS variant sequences for which RNAexpression ratios of the FS variant in tumor vs. normal cells weredetermined, confirming the differential expression.

TABLE 2b Gene Accession Name FS Peptides RNA NM 006306 SMC1L1GCCGIYCHEEPQREDSSI   98X (SEQ ID NO: 15) NM 015336 HIP 14 PWMCKKYYRLL   4X (SEQ ID NO: 16) XM 044434 KIAA1458 NPCQLLKPMVA  6.3X(SEQ ID NO: 17) NM_014342 MTCH2 SCWWMPSLLPNIYVLDRLL VHA   2.2XTKRGEYEPRK (SEQ ID NO: 18) NM 006833 COPS6 RGPL  9.75X (SEQ ID NO: 19)NM 000314 PTEN   41X

Table 2c shows FS variants having high odds ratios, computed asdescribed above; for CIAPIN1 and STYXL1, RNA expression ratios weremeasured to be 2.6× and 2.0×, respectively; RNA expression in tumorcells exceeding that in normal cells was confirmed by PCR and inspectionof electrophoresis gel band intensities in BCL2L12 and DNPEP; andexpression in tumor cells was verified by RNA extraction and sequencingfor BCL2L12, DNPEP, and STYXL1.

TABLE 2c Accession Gene Odds ID name FS peptide Ratio NM_001745 CAMLGVHICSISYFTTCVHGIIQIFSQE 2.50 (SEQ ID NO: 20) NM_001014438 CARSGSVHTSRWEKGDVVLLWANRL 2.86 (SEQ ID NO: 21) NM_020313 CIAPIN1SAHKESSFDIICQV 2.22 (SEQ ID NO: 22) NM 006716 DBF4 SS 4.09 NM 017996DET1 TRHLLKSMSTRAARQQRTYCRDT 2.67 KEKSCPMAMTSGQ (SEQ ID NO: 23)NM 012100 DNPEP GWLQ 2.36 (SEQ ID NO: 24) NM 006705 GADD45G LRGQGG 2.38(SEQ ID NO: 25) NM_000849 GSTM3 LLTMIEANGWM 3.82 (SEQ ID NO: 26)NM 201612 IKIP CGRNLKLSWNN 15.43 (SEQ ID NO: 27) NM_001012634 IL32HQAIERFYDKMLQNQDVDR 4.63 (SEQ ID NO: 28) NM_015416 LETMD1ESLEPGHASHILPASSLVETSFE 3.08 DSYNCDSPTGQGFGKAGDWPADC SGSKIGLLSPWPEFYAYW(SEQ ID NO: 29) NM_002405 MFNG GPTLWSPTAPRNTATQLCPARWL 2.47LSSTPSWPVGLGGSAMWTMTTM (SEQ ID NO: 30) NM_198883 MTX1KYNADYDLSARQGADTLAFMSLL 3.08 EEKLLPVL (SEQ ID NO: 31) NM_152298, NASPSNH 3.65 NM_002482 NM 006985 NPIP SRSQLGMAVIFLFTPR 2.11 (SEQ ID NO: 32)NM 153681 PIGP KNLKGSRVC 3.69 (SEQ ID NO: 33) NM 018845 RAG1API KLR 2.29NM 015014 RBM34 GKRSSEC 11.08 (SEQ ID NO: 34) NM 183400 RNF14AICSMQALRQPMGRTPWQRGPVC 11.73 LDAIS (SEQ ID NO: 35) NM_016211 SEC31APSEWLE 2.33 (SEQ ID NO: 36) NM_001009939 SEPT5- VENQAHCDFVKLRNMLIRTHMHD2.63 LKDVTCDVHYENYRAHCIQQMTS KLTQDSRMESPIPILPLPTPDAE T (SEQ ID NO: 37)NM_005827 SLC35B1 WWIVPGAGSMLPVLSPIWVPWSP 9.45 AIQHYSLSTTQLRSLVNPASQSQSCSLG (SEQ ID NO: 38) NM_003473 STAM GVILKYVKN 2.14 (SEQ ID NO: 39)NM 003763 STX16 A 2.70 NM_016086 STYXL1 GTGCISAIPH 7.27 (SEQ ID NO: 40)NM_032026 TATDN1 VYDYRWKSTRQ 3.63 (SEQ ID NO: 41) NM_001001563 TIMM50DHRAHQPLPSPRPSAGTVLPATL 2.92 HARFGAHRRPLAS (SEQ ID NO: 42) NM_100486 WACMEDKHSSDASSLLPQNILSQTSR 2.70 HNDRDYRLPRAETHSSSTPVQHPIKPVVHPTATPSTVPSSPFTLQS DHQPKKSFDANGASTLSKLPTP TSSVPAQKTERK(SEQ ID NO: 43) NM 024061 ZNF655 GHTSPPSHHPDS 2.09 (SEQ ID NO: 44)

Table 2d shows FS variant sequences for which sequence matches werefound in the tumor EST databases but for which, in the normal ESTdatabases, no sequence matches were found for either the FS variant orthe parent wild type sequence; this prevented computation of an oddsratio, but obviously nonexpression in normal cells is a desirablecharacteristic. Note that very short FS variant sequences arenevertheless significant since, when expressed, they result in peptidesrepresenting fusions of the FS variant with the adjacent unshiftedsequence.

TABLE 2d Gene Accession ID Name FS Peptides NM_212533 ABCA2 E NM_001606NM_172027 ABTB1 VLCLLVWARGAGTLPSGQWSPLRGQHLRW (SEQ ID NO: 45)NM_001033055 AIPL1 VIFHFRTMKCDEERTVIDDSRQVGQPMHIIIGNMFKLEVWEILLTSM RVH EVAEFWCDTI (SEQ ID NO: 46) NM_001707 BCL7BGQSLAMLSRLVVNSWPQAVPRP (SEQ ID NO: 47) NM_004328 BCS1L LES XM_043653BEXL1 PLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHS (SEQ ID NO: 48) NM_139343BIN1 LRKGPPVPPPPKHTPSKEVKQEQILSLFEDTFVPEISVTTPSQPAEASEVAGGTQPAAGAQEPGETAASEAAS (SEQ ID NO: 49) NM_015412 C3orf17 GNM_001009186 CCT6A QIQHPTASLIAKVATAQDDITGDGTTSNVLIIGELLKQADLYISE(SEQ ID NO: 50) NM_134445 CD99L2 QPWDHTNNHHNK (SEQ ID NO: 51) NM_033488CDC2L1 SVCTSPNDERGLQRQSESQPLESQPASAAAGAVRVGRRPEASKRRE NGRKGPAVRLTGHQRQREEDQLGRVLVSRIRLRF (SEQ ID NO: 52) NM_001005271 CHD3EMGEEGGGRTGNH NM_001005273 (SEQ ID NO: 53) NM_017828 COMMD4VRPSTVSMANPCPVNCSSWGCPKSTRPACAAVMRRSKAPCRSTC GSAAYA (SEQ ID NO: 54)NM_032179 CPSF3L SCLD (SEQ ID NO: 55) NM_004715 CTDP1KWTTSLEKAATTATARRGGLRSRRRSPSPGSQGPAGSGRSGHLRP ARGARQGAGGPEAT RGS(SEQ ID NO: 56) NM_001930 DHPS AERGRLRCLHQHSPGV (SEQ ID NO: 57)NM_182908 DHRS2 FHGNESLWKNFKEHHQLQRIGESEDCAGIVSFLCSPDASYVNGENI AVAGYSTRL(SEQ ID NO: 58) NM_021931 DHX35 HDLSSQRLQGE (SEQ ID NO: 59) NM_001009894DKFZp434 ISHTFGLD N2030 (SEQ ID NO: 60) NM_032378 EEF1DAQAPGPPAAPAETTVSSSSGLPVWKWRTRVCVAWYRSCSRPSPS WRPG (SEQ ID NO: 61)NM_024311 ET RRVTEEQCLLP (SEQ ID NO: 62) NM_023109 FGFR1CIHRDLAARNVLVTEDNVMKIADFGLARDIHHIDYY (SEQ ID NO: 63) NM_001001662FLJ16636 KDVGEPSLFPLA (SEQ ID NO: 64) NM 024578 FLJ22709VSLTGRGSPGRASRQKI (SEQ ID NO: 65) NM_005087 FXR1 GKRCD NM_001013439(SEQ ID NO: 66) NM_002106 H2AFZ VGI NM_014056 HIGD1AVFGDSPALSPRLECSGRISAHCSLCLLGSSDSPTSAS (SEQ ID NO: 67) NM_003529HIST1 H3A R NM_153490 KRT13 GPGPSR (SEQ ID NO: 68) NM 019016 KRT24ATPTWK (SEQ ID NO: 69) NM_015848 KRT2BTLLQEQGTKTVRQNLEPLFEQYINNLRRQLDNIVGERGRLDS (SEQ ID NO: 70) NM_002272KRT4 ESWYQTKYEELQITAGRHGDDLRNTKQEIAEINRMIQRLRSEIDHVKKQCANLQAAIADAEQRGEMALKDAKNKLE (SEQ ID NO: 71) NM_153486 LDHD GRRLR(SEQ ID NO: 72) XM_060417 LOCI 27295LARMCVPTLLLTNLRARLVRKREELSNVLAAMKKA TAKKD (SEQ ID NO: 73) XM_497978LOC132391 RVRHGVRGPGHRDSRGSGRNGRHPEREGDHAKPERPPGLLPGQ Q (SEQ ID NO: 74)XM_211339 LOC284120 LLSFCCPGWSSVA (SEQ ID NO: 75) XM_208312 LOC284120LDDSIVVKLVSPGSALPRIFGLSPESLSADH (SEQ ID NO: 76) XM_293903 LOC284120IVEERKMHWSPRTWSLGNQFMERRESRFRKEMTKLSTE (SEQ ID NO: 77) XM_370672LOC284120 TVKHPVCV (SEQ ID NO: 78) XM_495875 LOC284120FHVNHVKRSRVPLSVGDHTNSS (SEQ ID NO: 79) XM_372840 LOC391209LARMCVPTLLLTNLRARLVRKREELSNVLAAMKKATAKKD (SEQ ID NO: 80) XM_497922LOC391538 RCVLKIGEHTPSALAIMENAKCSGPLCQYLPAEWHCAHRGA (SEQ ID NO: 81)XM_496658 LOC440976 GGGGRAERPAGLAGVQGQTGWVSVLKPPALLPQLRSKVKRLIRF(SEQ ID NO: 82) XM_497335 LOC441632AKQVLLGRKVVVVRCEGINISGNFYTKQVEVPRFPPQADEHQLLPRLL PLPGPQPHLLADRARY AAPQDQARPGRSGPPQGV (SEQ ID NO: 83) XM_497347LOC441641 GNFYRNKLKYLAFLRKRMNTNPSRGPYHFRAPSRIFWRTVRGMLPHKTKRGQAALDRLKVFDGIPPPTT (SEQ ID NO: 84) XM_497605 LOC441836VGDEAQSKRGILTLKYPIEHGIVTTPSTTSCAWPRRSTRCC (SEQ ID NO: 85) XM_029323LOC90133 QAPRL (SEQ ID NO: 86) NM_138779 LOC93081GTCWRKWHRKCKLPIKSTGLRRQIIPWQ (SEQ ID NO: 87) NM_002383 MAZGFTTAAYLRIHAVKDHGLQAPRADRILCKLCSVHCKTPAQLAGHM QTHLGGAAPPVPGDAPQPQPTC(SEQ ID NO: 88) NM_174923 MGC31967 REEMSTQWLPTYVPIPPSCHKFPKNSQNHCSPHL(SEQ ID NO: 89) NM_182523 MGC61571 YFLSSIRFISTF (SEQ ID NO: 90)NM 025259 MSH5 RNPQQMPL (SEQ ID NO: 91) NM_002485 NBN V NM_001001716NFKBIB RHCTWL (SEQ ID NO: 92) NM_020729 0DF2L WRIFLH (SEQ ID NO: 93)NM_001007157 PHF14 GLADS (SEQ ID NO: 94) NM 015937 PIGTEFSSQLWTLKEGAEVAPGQ (SEQ ID NO: 95) NM_007221 PMF1SPLLHWDGSAWSPPALWWTVCETGLQLGGVQVTT GEEGGNL (SEQ ID NO: 96) NM_001017431RBM3 VVVVKDRETQRSRGFGFITFTNPDLWMVVRSVWIMQASLLGEPE EVALGPMGVVAATL(SEQ ID NO: 97) NM_015725 RDH8LFLWLSSQALTLRPCTTSGTSISQPPGSCFAPWDRTHRTWFRPLST SSARLDHPCADRPTSATRR(SEQ ID NO: 98) NM_194452 RNF121 IW NM 001005 RPS3 KLVGNSQKECGVS(SEQ ID NO: 99) NM_058192 RPUSD1GVSGVGGVLVVTEGKLRHRATKLMLGHPEHQGRAGNKHSCVLNSTPCSLSASHLTQGPCWLLTDSLGVWLAAILQDRAPPWPCPHQW (SEQ ID NO: 100) NM 207521RTN4 MDLKEQPGNTISAGQEDFPSVLLETAASL (SEQ ID NO: 101) NM_173073 SLC35C2RAALVLVVLLIAGGLFMFTYK (SEQ ID NO: 102) NM_130849 SLC39A4VRMARGGAALGRELSRGAEQGR (SEQ ID NO: 103) NM_003096 SNRPGKKLNGGRHVQGILRGFDPFMNLVIDECVEMATSGQQNNIGMVVI R GNSIIMLEALERV(SEQ ID NO: 104) NM_014748 SNX17 VGLAPLP (SEQ ID NO: 105) NM_013403STRN4 MLLRRRGTPSSPCARTTTAFVPWPSTTASRLCSPPPRTARSSSGTC RRRSRPRRMRR(SEQ ID NO: 106) NM 006521 TFE3 RGLQDPCHVVIFFIEGLAAAAANAGPGAGAGEA(SEQ ID NO: 107) NM_003299 TRA1 AWTRFAMRA (SEQ ID NO: 108) NM_176880TRA 16 VHRALRLSTRL (SEQ ID NO: 109) NM_173500 TTBK2GTKTCEAEPGAVVRAVHQQPQEAAGQHRGGTGSSGLGAEKHAG P GGGPQEQTMRMKSTSAQQQRMNL(SEQ ID NO: 110) NM_018299 UBE2W SCLLVKIFLFILMFIAMVISVYPF(SEQ ID NO: 111) NM 018206 VPS 3 5 SLIIIKRYGHF (SEQ ID NO: 112)NM_001006612 WBP5 A NM_001006614 NM_017528 WBSCR22 K NM_001033518 WIPI-2TRYGRCVHCREIVLQQPSGHRQP NM_001033519 (SEQ ID NO: 113) XM_374912 XRRA 1EDRKRGCCPTSSSLPISLRVRLS (SEQ ID NO: 114)

Table 2e shows FS variant sequences for which the number of sequencematches for the FS variant sequence against the normal EST databases waszero; this number was arbitrarily set at 1 for purposes of computing theodds ratio. RNA expression ratios measured for sequences C7orf24 andZWILCH were 3.6× and 10.4×, respectively; RNA expression in tumor cellsexceeding that in normal cells was confirmed by PCR and inspection ofelectrophoresis gel band intensities in DYRK4, HNRPUL1, MAP3K10, PPP4C,and RIPK2; and expression of the FS variant in tumor cells was confirmedby RNA extraction and sequencing for DYRK4, HNRPUL1, RIPK2, and ZWILCH.

TABLE 2e Gene Odds Accession ID name FS peptide Ratio NM_001033054 AIPL1HTGVYPILSRSLRQMAQGKDPTEWHVHTCGLANMFAY 3.00 HTLGYEDLDELQKEPQPLVFVIELLQ(SEQ ID NO: 115) NM_005787 ALG3 TQRLTGRPTWPR 2.79 (SEQ ID NO: 116)NM_001002857 ANXA2 VWMRSPLSTF 4.12 (SEQ ID NO: 117) NM_175073 APTXSLRKRQRTLAWKHTGRERDQATVIL 4.92 (SEQ ID NO: 118) NM_005174 ATP5C1CHQETKVHQKHPENYQVYENGSGSKICPS 2.62 (SEQ ID NO: 119) NM_001003785 ATP5HIFFFFGIHLGSIFILWHGNLQRIK 2.57 (SEQ ID NO: 120) NM_004047 ATP6V0B GS 2.13NM_080598 BAT1 GCCFFWWSVYQEG 2.12 (SEQ ID NO: 121) NM_013980 BNIP1SNQASWRKANLTCKIAIDNLEKAELLQGGDLLRQRPPK 5.33 RAWPRHPVPSLRASWGSAG(SEQ ID NO: 122) NM_018045 BSDC1 G 2.38 NM_001032363 C1orfI51 ESW 4.47NM_014145 C20orf30 APSCCQATSAKGGQTGPFQC 6.25 (SEQ ID NO: 123) NM_004649C21orf33 DPGAPEPWRG 2.31 (SEQ ID NO: 124) NM_005768 C3F AERE 3.00(SEQ ID NO: 125) NM_024051 C7orf24 ARRG 5.00 (SEQ ID NO: 126) NM_018491CBWD1 VIQRLLC 3.04 (SEQ ID NO: 127) NM_018246 CCDC25 GKNCDSGEESK 3.00(SEQ ID NO: 128) NM_001782 CD72 RPRG 5.83 (SEQ ID NO: 129) NM_006319CDIPT AACWTLSMDTLLALLIKEPGLGPCWTC 2.11 (SEQ ID NO: 130) NM_024300 CHCHD7CRSCSTF 6.55 (SEQ ID NO: 131) NM_001009566 CLSTN1 GERRE 3.79(SEQ ID NO: 132) NM_199442 COPE RDSIVAELDREMSRSVDVTNTTFLLMAASIYLHDQNPD3.30 AALRALHQGDSLE (SEQ ID NO: 133) NM_032589 DSCR8 LQTLEIKKVLE 7.00(SEQ ID NO: 134) NM_020185 DUSP22 DKTFQRKY 5.00 (SEQ ID NO: 135)NM_003845 DYRK4 IPKVFLKI 7.33 (SEQ ID NO: 136) NM_001967 EIF4A2DPKGNSGTWRLYGSHLSCLHWWNKCSK 5.62 (SEQ ID NO: 137) NM_019002 ETAA16KFKFECNFRSYEYRNYYL 3.00 (SEQ ID NO: 138) NM_032231 FAM96A VGNLHF 4.26(SEQ ID NO: 139) NM_005687 FARSLB NI 5.28 NM_001031704 FLJ20211GCQPDHGAGAWAACVP 2.00 (SEQ ID NO: 140) NM_013393 FTSJ2 IPALLLASCLG 2.22(SEQ ID NO: 141) NM_203504 G3BP2 EL 2.82 NM_004127 GPS1SCRTHPTPSLRAAWSPQPWTRPGWRPRGRRRC 6.31 (SEQ ID NO: 142) NM_012203 GRHPRAVRWSSGTRMSPSLPRS 17.27 (SEQ ID NO: 143) NM_147149 GSTM4LPYLIDGAHKITQSNAILCYIARKHNLCGETEEEKIRVDI 2.41 LENQAMDVSNQLARVCYSPDFEKL(SEQ ID NO: 144) NM_000853 GSTT1 VWPSCST 6.32 (SEQ ID NO: 145) NM_145871GSTZ1 LAIIEYLEEMRPTPRLLPQDPKKRASVRMISDLIAGGIQP LQ 7.31 (SEQ ID NO: 146)NM_000858 GUK1 GR 2.68 NM_000187 HGD GTA 3.33 NM_003537 HIST1H3B RW 2.15NM_001002032 HN1 GEGDIHENVDTDLPGSLGQSEEKPVPAAPVPSPVAPAPV 45.29PSRRNPPGGKSSLVLG (SEQ ID NO: 147) NM_144733 HNRPUL1MPWTILPGRTNSTIPKSSNKKTSQATRGDHWKWSSSRPI 2.40 VQK (SEQ ID NO: 148)NM_016371 HSD17B7 MIKKWLYVICVEDHVSEIRLYISKCWDHA 2.77 (SEQ ID NO: 149)NM_144981 IMMP1L CQWVMFG 2.00 (SEQ ID NO: 150) NM_024710 ISOC2EHDPGPPRPGAAGPCGGGRLLLTQPGGPAGGSGPHETE 8.85 W CLPLHQRR AHS A ACGRCRPPP VQGDPETHQGARPRQ RTAGPLPRPELPPPL (SEQ ID NO: 151) NM_005886 KATNB1 A 3.50XM_371877 K1AA0960 RKAQRYTGQ 5.00 (SEQ ID NO: 152) NM_138787 LOC119710DLLLLPGEVEQDV STSIPSCIPFV AQPPTCEVKPKPS VKR 5.25MDKQTEEILGDEVQLFSLDEEFDYDNVMLTSKFSPAEIE NIKELCKQQKRKDTSPDLEKSCD(SEQ ID NO: 153) XM_059341 LOC129293 GSSLL 2.50 (SEQ ID NO: 154)NM_001031744 LOC158160 MIKKWLYVICVEDHVSEIRPYISKCWDHA 3.64(SEQ ID NO: 155) NM_174928 LOC221143 VPSWKNRQQNSLE 4.44 (SEQ ID NO: 156)XM_290671 LOC339047 CKTWHSAWV 4.36 (SEQ ID NO: 157) NM_001005920LOC339123 VSACPSVPGHSRPCWARPLSPLPAPAEVPGPVLPRQVAGF 6.67VWGQSGPAEHRQHLLLPQSGLALPGVCGAAAAPPGPHLP GQ (SEQ ID NO: 158) XM_292085LOC341457 MPSTASPW AASPLSCLQTSFQRQQETFML 7.66 (SEQ ID NO: 159) XM_495885LOC440055 YVYQSQYCGFLQPEQNCHPREEGMEFMVLAQKF 6.32 (SEQ ID NO: 160)XM_352159 LOC440341 CKTWHSAWV 2.55 (SEQ ID NO: 161) NM_002446 MAP3K10RMLGPRPPRAARFR 4.80 (SEQ ID NO: 162) NM_181514 MRPL21 G 2.86 NM_145330MRPL33 KNILVRMVSEAGTGFCFNTKRNRLREKLTLLHYDPVVK 17.50 QRVLFVEKKKIRSL(SEQ ID NO: 163) NM_012333 MYCBP SVGSLI 3.78 (SEQ ID NO: 164) NM_012225NUBP2 R 7.14 NM_000430 PAFAH1B1 MKN 2.17 NM_001003 891 PCQAPRGCHEESWCGTQ 5.60 (SEQ ID NO: 165) NM_020992 PDL1M1 I 2.77 NM_002677PMP2 EVGVGLPPGKWLAWPNLT 4.50 (SEQ ID NO: 166) NM_174930 PMS2L5 LFQL 2.12(SEQ ID NO: 167) NM_006243 PPP2R5A KLYCSF 2.00 (SEQ ID NO: 168)NM_180977 PPP2R5D LFLIH 3.11 (SEQ ID NO: 169) NM_002720 PPP4CRCAATSMDNSMTSKSCSE 3.81 (SEQ ID NO: 170) NM_032864 PRPF38A RNAMY 4.44(SEQ ID NO: 171) NM_002767 PRPSAP2 ENKSTNSRVCEGKRCFHHPNCFEGREHHHHGAPDHGV7.38 CM (SEQ ID NO: 172) NM_021222 PRUNE K 2.77 NM_003579 RAD54L DA 2.43NM_005493 RANBP9 AKFVSYCGASNTRRSGRCQFWATSFRV 3.00 (SEQ ID NO: 173) NM_006743 RBM3 VSGWSSDPCGSCRQV CSGNQRRWLWGPWAWSQLL 3.81 (SEQ ID NO: 174)NM_181471 RFC2 GH 3.28 NM_003821 RIPK2 RKLNILMLLGH 4.80 (SEQ ID NO: 175)NM_001016 RPS12 YVYQSQYCGFLQPEQNCHPREEGMEFMVLAQKF 6.21 (SEQ ID NO: 176)NM_007008 RTN4 GFVFAPR 6.68 (SEQ ID NO: 177) NM_005888 SLC25A3YSCEFGSAKYYALCGFGGVLSCGLTHTAVVPLDLV 2.38 (SEQ ID NO: 178) NM_003136SRP54 VCY 3.38 NM_139276 STAT3 FIDAVWK 2.31 (SEQ ID NO: 179) NM_003195TCEA2 RLSPSVSHSICRRQQFGV 4.00 (SEQ ID NO: 180) NM_144582 TEX261 D 2.14NM_005727 TSPAN1 VCETQLHRLMTKSPLAFDTRPWDSQTLLWTPLGSGFCL 4.91TFPGGGLGQGGHEGLSLPKTQTPVPHSVLLHPPPHLHC (SEQ ID NO: 181) NM_018943 TUBA8MRECISVHVGQAGV 2.85 (SEQ ID NO: 182) NM_145345 UBXD5EDEVDMLSDGCGSEERRSQSLPAMAA 3.67 (SEQ ID NO: 183) NM_005153 USP10DKNIRELSLVSMKSLNPVTLCREPPATVFQAH 2.07 (SEQ ID NO: 184) NM_022170 WBSCR1GFRDDFLGGRGGSRPGDRRTGPPMGSRFRDGPPLRGSN 2.55MDFREPTEEERAQRPRLQLKPRTVATPLNQVANPNSAIFG GARPREEVVQKEQE (SEQ ID NO: 185)NM_024699 ZFAND1 IFFHLCVMIVQEYF 2.81 (SEQ ID NO: 186) NM_017975 ZWILCHCPAEIK 4.42 (SEQ ID NO: 187)

Table 2f shows FS variant sequences for which computed odds ratios wereless than 2.0, but which are likely to be involved in tumorigenesis; RNAexpression of BCL2L13 and DTYMK in tumor cells exceeding that in normalcells was confirmed by PCR and inspection of electrophoresis gel bandintensities, and expression of DTYMK in tumor cells was confirmed by RNAextraction and sequencing.

TABLE 2f Acces- sion Gene ID Name Function FS Peptides NM_ BCL2L13 Apop-QFWCLWFCYDKCFWN 015367 tosis (SEQ ID NO: 188) NM_ DTYMK Kinase IFP012145 NM_ PSMA7 ETC RYTQSNGRRPFGISALIVGFDFDGTPR 152255LYQTDPSGTYHAWKANAIGRGAKSVRE FLEKNYTDEAIETDDLTIKLVIKALLEVVQSGGKNIELAVMRRDQSLKILNPEE IEKYVAEIEKEKEENEKKKQKKAS (SEQ ID NO: 189)NM_ GALNT14 ETC KYGPSHTPSRSSRRSCACQSSPCSLAP 024572QWFLSFARMEMTDSNGPKLVPTSST (SEQ ID NO: 190) NM_ SNRP70 ETC RPGPGP 003089(SEQ ID NO: 191)

Table 2g shows genes for which FS variants were predicted and oddsratios computed as shown, but whose variants arise from indels of lessthan 10 bp, increasing the likelihood that the difference are due to asequencing error.

TABLE 2g Accession Odds ID Definition Ratio FS Peptides XP_ PREDICTED:53.19 VSELACIYSASFCTTMR 060328.1-11 similar to  (SEQ ID NO: 192)60S acidic NP_ CD69 anti- 48 VQANTHSQCHQTAMFLH 001772.1-67 gen (p60,ALRTGLATRGNATLFLL early  (SEQ ID NO: 193) T-cell) NP_ ribosomal  41.66SPRSWAGPVLRDSARRC 001022.1-13 protein AWNSWTTRADPSSAM S28 [Homo(SEQ ID NO: 194) sapiens] XP_ PREDICTED: 33.65 ATSTLGASSAM 060328.1-51similar  (SEQ ID NO: 195) to 60S acidic  ribosomal protein P2 NP_ [Homo 22.47 VLASLPVYLLVGL 000995.1-62 sapiens] (SEQ ID NO: 196) NP_ ribosomal 20.69 VLALVVSVQTGTV 001025172.1- protein (SEQ ID NO: 197) 16 S29 isoform 2 XP_ PREDICTED: 18.67 VSDGVIKGVQRHEGA 497649.1-13 similar (SEQ ID NO: 198) to  Cofilin, NP_ alpha 2  16 VHQGPCWPPWSPWPSWT000080.2-10 type I SRCKRWWL 80 collagen  (SEQ ID NO: 199) (Homo XP_PREDICTED: 14.36 WAASPLSCLQTRSQRQQ 170597.1-16 similar  KIFVL to A TP(SEQ ID NO: 200) synthase,  H+ NP_ trans- 14.14 LGASSLVMPGTLL005167.1-74 porting, (SEQ ID NO: 201) murine mammary NP_ tumor  13.52WTFLVIPTW 001559.1-45 inte- (SEQ ID NO: 202) gration NP_ hemato- 12.8CGLQVVDPIFH 01002032.1- logical (SEQ ID NO: 203) 20 and  neuro- logicalNP_ reticu- 12.5 LQVDVGIYLCWCLV 722550.1-28  lon 4 (SEQ ID NO: 204) 6isoform  B [Homo NP_ RNA  12.48 VLVSSPSPTQSMLQLP 01017430.1- binding(SEQ ID NO: 205) 47 motif- protein  3 cal- nexin precur- sor NP_ [Homo 11.66 MLRLMMDMMMM 001737.1-18 sapiens] (SEQ ID NO: 206) NP_ reticu-10.71 LQVDVGIYLCWCLV 008939.1-11  lon 4 (SEQ ID NO: 207) 2 isoform C [Homo NP_ RNA  10.70 LVSSPSPTQSMLQLP 001017430.1- binding(SEQ ID NO: 208) 48 motif  protein  3 NP_ Nucleo- 10.28 LEVVARFHRKK954654.1-14 phos-min (SEQ ID NO: 209) 4 1 iso- form 2 [Homo NP_ general10.2 WLMSSRSEWVN 97001.1-48 trans- (SEQ ID NO: 210) cription factor IIH, NP_ protea- 10.11 VIQRPAATLRTTWALSH 002801.1-36 some 26S WLMTVKCnon- (SEQ ID NO: 211) ATPase subunit  4 NP_ 15 kDa 10.09 VDENWEGSLKSKLC004252.2-93 seleno- (SEQ ID NO: 212) protein isoform 1 XP_ PREDICTED: 10CEYSTPTSMGGGK 371019.1-12 similar to (SEQ ID NO: 213) ribosomal

Example 6

FIGS. 7 a, 7 b, and 7 c illustrate examples of a method for assessingthe likely utility of a predicted candidate novopeptide as a cancervaccine component by comparing the RNA expression level of transcriptscontaining the sequence of the novopeptide in tumor cells with that innon-cancerous cells. FIG. 7a demonstrates amplification of a FS variantin BCL2L13 cDNA from three different human tumor cell lines, but notcDNA obtained from normal tissue. PCR primers were designed such thatthey flanked the BCL2L13 FS region and amplify a FS of 253 bp ifpresent. The left half of the figure shows amplification of threedifferent human tumor cDNA preparations. Lane labels in FIG. 7 are asfollows. Lane M, 100 bp molecular weight marker; Lane 1, MCF-7 humanbreast cancer cell line; Lane 2, SW480 human colon cancer cell line;Lane 3, DU-145, human prostate cancer cell line; Lane TA beta actin fromSW480 cell line. Right side of gel: Lane NA, beta actin from normalcolon; Lane NL, normal lung; NB, normal breast; NC normal colon.

FIGS. 7b and 7c show two additional examples of amplification of cDNAfrom transcripts containing sequence corresponding to frameshiftednovopeptides relating to genes designated STYXL1 and HNRPUL1. Theagarose gel shows a frameshift encoding a novopeptide present in tumorcells, but not present in cDNA from normal lung, breast and colon. PCRwas performed as in 7a, but with primers that flank the predictedframeshifts. Lanes are the same as FIG. 7 a. Arrows mark the FS bands ineach figure.

Example 7

Quantitative PCR measurement showed over-expression in tumor samples oftranscripts containing sequence corresponding to another frameshiftvariant, SMC-1A cDNA from four fresh human pancreas tumor samples weretested for relative expression message containing sequence correspondingto SMC-1A FS using PCR primers specific for the FS sequence. Levels ofFS SMC-1A cDNA were compared to SMC-1A cDNA amplified from normalpancreas from the same patient. Table 3 shows that three of fourpancreas tumors overexpressed FS SMC-1A, compared to the normal wildtype sequence.

TABLE 3 Relative Expression Level Sample FS WT Panc-C 2.69 0.094 Panc-E30.7 0.852 Panc-F 1.15 0.26 Panc-G 0.512 0.696

Example 8

This example shows that novopeptides actually expressed by tumor cellscan be identified via mass spectrometry, and discloses a method fordoing so, and also illustrates and discloses a method for identifyingsubsequences likely to be displayed in MHC. Peptides were eluted fromthe surface of tumor cells by exposure to 100 mM citric acid for 30seconds, or phosphate buffered saline for 4 hours, or peptides werecompeted from cell surface HLA molecules with a biotinylated peptidehaving high affinity for the HLA molecule of interest. A database offrame shifted peptide sequences was constructed from the sequencespredicted bioinformatically as described above, to enable the use ofLC-MS/MS to identify novopeptides actually present in the eluted sample.The peptide sequence database was used to search spectra obtained fromLC-MS/MS, using Spectrum Mill, for peptides eluted from MCF-7 breasttumor cells HLA-A*0201,-B* 18/44 and -Cw*05. The HLA types weredetermined for the tumor cells of interest as described above.Unexpectedly, peptides longer than 8-10 amino acids were identified fromLC-MS/MS analysis of the elutions that matched some sequences in the FSdatabase. These longer peptides have been analyzed using MHC class Ibinding algorithms, BIMAS and SYFPEITHI, to identify preferred 9-mersequences that are capable of binding multiple HLA class I molecules asshown below in Table 4a-4e. The algorithms use different methods ofscoring peptides for binding. Sometimes the algorithms arecomplementary, but often they are not. BIMAS values over 150 andSYFPEITHI values over 20 have the best chance for peptides binding toMHC intracellularly and being transported to the cell surface.

TABLE 4a Parent sequence #1 eluted from MCF-7 tumor cells:VIKSLQSWYLRLVI (SEQ ID NO: 225) HLA SEQ BIMASS SYFPEITHI A*0201SLQSWYLRL 32 23 (SEQ ID NO: 214) A*1101 KSLQSWYLR .036 21(SEQ ID NO: 215)

TABLE 4b Parent sequence #2 eluted from MCF-7 tumor cells:FLSPMSGLLSTTQQSACTGIHRTS (SEQ ID NO: 226) HLA SEQ BIMASS SYFPEITHIA*0201 FLSPMSGLL 12.7 23 (SEQ ID NO: 216) A*1101 QSACTGIHR 0.008 23(SEQ ID NO: 217) A*6801 QSACTGIHR 45 20 (SEQ ID NO 217)

TABLE 4c Parent sequence #3 eluted from MCF-7 tumor cells:PSPQETEFPGPGVVRPILDVGKIS (SEQ ID NO: 227) HLA SEQ BIMASS SYFPEITHIA*0201 GVVRPILDV 13 21 (SEQ ID NO: 218) A*1301 VVRPILDVG 0.405 20(SEQ ID NO: 219) B*0702 GPGVVRPIL 120 23 (SEQ ID NO: 220) B*2705VRPILDVGK 2000 23 (SEQ ID NO: 221) B*5101 RPILDVGKI 200 24(SEQ ID NO: 222) B*5102 RPILDVGKI 2640 NA (SEQ ID NO: 222)

TABLE 4d Parent sequence #4 eluted from MCF-7 tumor cells:GQDCYRVPVTED (SEQ ID NO: 228) NO HLA matches

TABLE 4e Parent sequence #5 eluted from MCF-7 tumor cells:AGLGTKLAAEGLAPN (SEQ ID NO: 229) HLA SEQ BIMASS SYFPEITHI A*0301KLAAEGLAP 0.120 23 (SEQ ID NO: 223) A*0801 GTKLAAEGL 4.0 22(SEQ ID NO: 224)

In related LC-MS/MS experiments, a 9-mer peptide bioinformaticallypredicted to be a FS of the BCL2L13 gene was identified in LC-MS/MSspectra from MCF-7 breast tumor cell elution experiments using themethods described above. The sequence of this peptide is CLWFCYDKC (SEQID NO: 230) and fits the HLA-A*0201 binding motif.

Example 9

Novopeptides in the FS database in association with particular tumorcell types were identified. As described in Example 8, it was observedthat peptides longer than 8-10 amino acids (the expected size for MHCelutions) were obtained that matched FS sequences in the FS database.Typically peptides longer than 8-10 amino acids form epitopes forantibodies. Pursuant to the current teaching that protective ortherapeutic antibodies may be generated to FS after vaccination, serumtaken from patients with different tumor types was assayed forreactivity with predicted novopeptides by standard ELISA techniques.FIG. 8 shows one cancer patient in 23 with antibody reactivity in serato peptide FS 6-21 sequence. This finding reveals novopeptides thatelicit an anti-tumor antibody response upon vaccination with saidnovopeptides. Reactive sera 801 is indicated.

Example 10

FIG. 9 shows that the probable immunoprotectiveness of a predictednovopeptide can be assayed by immunological screening via a CTL assay,and discloses one method for doing so. CTLs activated againstnovopeptide FS 6-21, described above were able to kill MHC-matched tumorcells pulsed with FS 6-21 peptide 901, but not unpulsed SW480 tumorcells 903 as shown by the square symbol. Since SW480 tumor cells do notexpress FS 6-21 novopeptide endogenously, the cells required peptidepulsing. This is a standard Cr release assay that anyone skilled in theart would be able to do.

Example 11

A predicted novopeptide elicits a strong antibody response by geneticvaccination. A method for assay of this response is shown. Mice wereimmunized as described above with a gene vaccine encoding FS 6-21novopeptide. Serum was obtained from the mice and incubated with B16tumor cells. Antibodies specific to novopeptide FS 6-21 were shown tospecifically bind B16 murine tumor cells, while pre-immune sera did notbind. FIG. 10 shows immunofluorescence images demonstrating that theanti-FS 6-21 serum binds specifically to B16 tumor cells and 4T1 breasttumor line cells and the pre-immune serum does not.

Example 12

Novopeptides can also confer therapeutic as well as prophylacticprotection. Mice were injected with the B16 tumor cells and then one daylater immunized with the FS 1-78 and FS 6-21 novopeptides as genevaccines. As shown in FIG. 11, the animals receiving both peptides wereprotected relative to the control animals, but this protection is not asstrong as a prophylactic vaccination, as indicated by the lower survivalrate (one-third of the mice survived, triangle symbol, compared to 80percent survival shown in FIG. 6 for prophylactic immunization). On Day0, mice were injected with 10⁵ tumor cells. One day later, mice werevaccinated with the FS 6-21 peptide sequence 1103, the FS1-78 peptidesequences 1105, a combination of both 1101, or an irrelevant peptidesequence 1107.

Example 13

Candidate novopeptides that are capable of being displayed only in oneor a few HLA types that are poorly represented in the target populationare less desirable than those capable of being displayed in multiple HLAtypes that are shared by larger segments of the target population. Here,tumor targets of interest were HLA typed, with the results as shown inTable 5, so that bioinformatically identified candidate novopeptides canthen be screened using MHC class I binding algorithms, such as, forexample, BIMAS and SYFPEITHI, to determine the novopeptide sequencesmost likely to be capable of being displayed on the MHC types present inthe tumors of interest. This information was used to determine the HLAtypes for purposes of LC-MS/MS identification and sequencing asdescribed above.

TABLE 5 Tumor cell line Histological type HLA type MCF-7 Breast(epithelial A*02/02, B* 18/44, Cw*05/05 adenocarinoma) SW480 Colon(adenocarcinoma) A*02/24, B*07/15, Cw*07/07 A549 Lung (carcinoma)A*25/30, B* 18/44, Cw* 12/16 Panc-1 Pancreas (epithelioid A*02/I 1,B*38/38, Cw*12/12 carcinoma) DU-145 Prostate (carcinoma) A*03/33,B*50/57, Cw*06/06

Example 14

A novopeptide associated mutation was identified that occurs in alltumors of humans and mouse identified in the public databases. Thiscomprised a frameshift and the frameshifted gene encodes the SMC1A geneand has the sequence NGSGCSGVYCHEEPQGEDSSV (SEQ ID NO: 8) as compared tothe normal wild type sequence of: NGSGKSNVMDALSFVMGEKIAN (SEQ ID NO: 7).The frameshift was found through informatic analysis of human cancercDNA sequences compared to normal tissue. Public databases were used.The presence of the same FS was determined in mouse breast and melanomatumor lines by sequencing cDNA from these tumor lines in the homolog ofthe SMC1A gene. Thirty-one (31) human tumor libraries were examined forthe presence of the FS. In all 30 that were sequenced, the FS in SMC 1Awas identified as appearing in all lung, breast and melanoma samples butnot normal samples. This correlation indicates that this mutation isoncogenic. This frameshift mutation can be used alone or in combinationas a component or entirety of a vaccine, either therapeutic orprophylactic, against cancer. It can also be used diagnostically todetect early cancers. This mutation creates an oncogene that is a newanti-cancer drug target.

The FS was tested for therapeutic value as a vaccine in the mouse tumormodel. The B16 melanoma line was inoculated into mice. One day later themice were vaccinated with a gene vaccine encoding the FS. As shown inFIG. 12 a, no therapeutic effect on tumor size progression was noted forthe SMC-1 FS 1201 relative to the control 1203. However, the 17aanovopeptide is predicted to bind the mouse B16 MHC class I molecules(MHCI) poorly. Therefore epitope-enhanced variants were made based onpublic programs for improving MHCI binding. When these mice weretherapeutically vaccinated using the epitope enhanced novopeptide 1205there was a positive effect. Similarly, as shown in FIG. 12 b, thenon-epitope enhanced novopeptide 1201 did not show significantimprovement in survival time relative to the control 1203, but theepitope enhanced variant 1205 did show an improvement. Therefore the FSis present specifically in human and mouse tumors and is therapeuticwhen epitope enhanced for mouse. Epitope enhancement is not necessaryfor human tumors.

Example 15

An example embodiment of a method for producing vaccine components asdisclosed herein is as follows:

(a) The NCI database and/or data from the Cancer Genome Atlas issearched and analyzed to find either a variant in the cDNA (RNA) oftumors or in the genomic DNA of tumors that is rare or absent in normalsequences of cDNA (RNA) or DNA.

(b) The presence of the variant in cDNA or mutation in DNA is confirmedby sequencing the cDNA or DNA of tumor cells and normal cells andcomparing the two. A panel of tumor and normal cell cDNA and DNA areused to obtain an initial estimate of the frequency of the mutation orvariation in tumors versus normal cells.

(c) RNA is extracted from tumor and normal cells and the relativeexpression of normal messenger RNA versus the RNA encoding thenovopeptide is estimated by PCR. In the case of a mutation noted in theDNA, if the mRNA of that mutation is not present in the tumors, thecorresponding novopeptide is not pursued. In the case of a variant inRNA, if the variant RNA is present at nearly the same level in tumors asnormal cells, the corresponding novopeptide is not pursued.

(d) Candidates remaining at this point may be screened by massspectrometry for being present as novopeptides on the surface of tumorcells but not normal cells. The preference is to elute peptides by acidelution or incubation in buffer to collect the medium. While peptidesbinding the MHCI can be eluted by more specific protocols, these wouldmiss non MHCI peptides that may be targets of anti-tumor antibodiesgenerated by the vaccine. Chromatographs from the mass spectrometry arecompared to the unique database of possible frameshift novopeptidesdescribed herein. Candidate novopeptides may be confirmed by massspectrometry sequencing. If a novopeptide is discovered which is longerthan 9 amino acids, the MHCI eluted peptides may be specificallyanalyzed for the presence of a nested peptide sequence that would bepredicted to bind the HLA of the particular tumor cell.

(e) The candidate novopeptide sequences or nested subsequences thereofmay be screened for predicted binding to human MHCI molecules. Thosethat are predicted to bind tightly to common MHCIs receive relativelyhigher scores than those predicted to bind weakly, or to bind stronglyto rare MHCIs.

(f) For those peptides that receive relatively high scores, mouse tumorcells may be assayed by PCR or sequencing for the presence of the RNAencoding the novopeptide. If present in mouse tumors, mice may bevaccinated with the novopeptide and then challenged with tumor cells todetermine if the peptide is protective.

(g) In parallel the high scoring candidates may be screened for theirpresence in human tumors. A number of tumors of the same type as well asa number of different tumors are screened by PCR or sequencing of RNA todetermine the overall frequency in patients. In parallel a large numberof cell types and cell types from a large number of normal subjects maybe screened in the same fashion for the presence of the novopeptideencoding RNA. Novopeptide variants that are very infrequent or at verylow levels in normal RNA and are present at higher levels in at least10% of tumors of one type or in 10% in all tumor types proceed to thefinal screen.

(h) A final defining screen may be employed to determine if thecandidate is useful in a prophylactic vaccine. This step may be requiredbefore testing of a vaccine in human subjects would be permitted orappropriate. The first screen is for T-cell reactivity. T-cells fromhumans with the relevant MHCIs will be activated with the testnovopeptide. Once a population of such T-cells are created they will bereacted with human tumor cells with the corresponding MHCI type. Ifthese tumor cells are killed or inhibited in growth, such would confirmthat these tumor cells are presenting the novopeptide and aresusceptible to the vaccine. This same T-cell preparation will also bereacted against a panel of normal cells of the same MHCI. If these cellsare not killed or growth inhibited by the novopeptide activated T-cells,such would confirm that this novopeptide is a validated candidate for acancer vaccine.

(i) For antibody screening the process is simpler. Antibody will begenerated to the test novopeptide. This antibody will be reacted againsttumor cells to determine if it binds to the surface. If it does thisindicates that the tumor is susceptible to the antibody. As above, thesame antibody will be reacted to a panel of human normal cells.Novopeptides that induce antibody specific binding are validated ascomponents of a prophylactic cancer vaccine.

Example 16

Novopeptides useful (in their entirety, or operable portions orequivalents thereof) as peptide components of an anti-cancer vaccine aredisclosed in Tables 6, 7, and 8 and were identified as follows:Bioinformatic screening was performed to identify chimeric transcriptsin the NCBI EST sequence database by screening for transcriptscontaining a continuous sequence that aligns to two distinct RefSeqsequences simultaneously, and that, on translation, would produce aframeshift relative to wild type in the portion corresponding to thedownstream RefSeq. Approximately 8 million sequences were screened,resulting in approximately 5,000 candidates. From this candidate set,candidates were excluded if a functional transcriptional coding sequencewas not present in the upstream portion; if the chimeric EST sequencewas not present in at least two independent cDNA libraries, or in threeor more copies in one library, or had a junction point at an exonboundary of both portions; if the upstream portion corresponded to areverse strand; or if the candidate was too short (less than 6 residues,or otherwise too short to accommodate primers for PCR verification). Theremaining 233 candidates were screened against 50 breast cancer lines byRTPCR using primer pairs designed to amplify the predicted chimericsequences. Of the candidates screened, 48 were verified as present incDNA of the cancer lines. Table 6 shows these validated sequences, inwhich the first 10 residues are the 10 residues of the upstream sequenceimmediately adjacent to the junction point, read in their wild typereading frame, and the remaining residues are the downstream sequencefrom the junction point to the first stop codon, read in thenon-wild-type reading frame arising from the out of frame junction. Alsoshown are the two reference exons to which the two conjoined domainsalign.

TABLE 6 SEQ ID REFERENCE EXONS NOVOPEPTIDE SEQUENCE SEQ IDTHAP2_Exon2_TME FDFCTHIKSMVTYDLFLRGVGCFLLLFLF NO: 240 M19_Exon3 SEQ IDBOLA2_Exon2_SMG EGKPLLQRHRLLNR NO: 243 1_Exon12 SEQ ID GFOD1_Exon1_C6orLTRQIAVKTLEPGHQRKKISRQKNTGEKKMPRGSVQLSFCSLQHPH NO: 244 f114_Exon2MGHLFTPHDAALGESQGTGFKPLGMQPV SEQ ID MDS1_Exon2_EVI1_NLKDPSYGWEILDEFYNVKFCIDASQPDVGSWLKYIRFAGCYDQHN NO: 245 Exon4LVACQINDQIFYRVVADIAPGEELLLFMKSEDYPHETMAPDIHEERQYRCEDCDQLFESKAELADHQKFPCSTPHSAFSMVEEDFQQKLESENDLQEIHTIQECKECDQVFPDLQSLEKHMLSHTEEREYKCDQCPKAFNWKSNLIRHQMSHDSGKHYECENCAKVFTDPSNLQRHIRSQHVGARAHACPECGKTFATSSGLKQHKHIHSSVKPFICEV SEQ ID C11orf79_Exon3_C1NDWDIYYWATGPEGPFRHPGARASGHHGAGAQGSASAPPAAGP NO: 246 1orf66_Exon5GPAGAGELPTWPTLHDVGVQFQVSQGPSRPARFLAEEIDRRKGGEWLHQTVPPEPHCLPTALTGPPWGPCPPPRPECHQVRLPPQDSPT WR SEQ ID ABHD14A_Exon3_AGYRAVALDLPAHHAQRHDQQGSRGGAPIGDALPPVPAYPHCPAQ NO: 247 CY1_Exon2 A SEQ IDRBM14_NA_RBM4_ GRVIECDVVKGSCQDGEAVHRKPAPGGYRAGDSLTLRAVWEGAG NO: 248Exon2 M SEQ ID C20orf29_Exon2_VI LRKEQILAVASLVSSQSIHPSWGQSPLSRI NO: 249SA_Exon2 SEQ ID RRM2_Exon9_C2orfLMLELGFSKVLGDREVQSRWSPGPRGDSTPVREMETNHPPSVRG NO: 250 48_Exon2 SEQ IDELAC1_Exon2_SMA QLMKSQLKAGYPEYMSNNFPCNVSCCFSLFPKDQNCFRNWRHI NO: 251D4_Exon2 SEQ ID BCAS4_Exon1_BCAS LFLTPEPGAEVPLTGA NO: 252 3_Exon24SEQ ID C22orf39_Exon2_HI WEERRNAEAQASRFFQLIFTLTGPSSQLEDKGRILGRL NO: 253RA_Exon2 SEQ ID PMF1_Exon4_BGLA QVQAQQQAWQVRSPAVQSPAKVQPLCPSRRAARNO: 254 P_Exon4 SEQ ID SDHD_Exon3_TEX12LAAALTLHGHCLQCQIVHSCPLLENQIHLSLKFPDYFIKMKPWRKI NO: 255 _Exon3 SEQ IDPRR13_Exon3b_PCB KHHKYHKHGKFLAFTPNQ NO: 256 P2_Exon2 SEQ IDRMND5A_Exon2_AN VSRVGKAIDKDSL NO: 257 APC1_Exon25 SEQ ID TYMP_Exon9_SCO2VDVGQRLRRGASDPCCC NO: 258 _Exon2 SEQ ID NAIP_Exon13_OCLN ISAEYDPSKLGNO: 259 _Exon5 SEQ ID C1orf151_Exon1_NB CLADAVVKIGLWRPRA NO: 260L1_Exon3 SEQ ID DDIT3_{circumflex over ( )}Exon3_MARENGGTYVSPPLPLGASGGFPSATANCFFRSKSFATSAATSFLSAFCAF NO: 261 S_{circumflexover ( )}Exon21 SSRTMFPCFVTSSISACICCGLAVVTVSTTAGFGDVFAWPPPKRCLKLSIWSFSNFWNKGLTVPIWCPAGKVHRKFVSRILQAGGGSCSWAW IVALTVGM SEQ IDRIPK3_Exon9_ADCY RTPEPNPVTGADLRPELPDHCAVRAGRLLAAAGPRFPGAATAALDA NO: 2624_Exon2 SPVRLGMGRAASARPRLPVHRGRGERLGPGVLFSLRHLHGVCHAALGHAGRRRRGPRLLTLASAGPRAVSWATAGLTACTAAAVGSKRSAVPVRERGRSVPQGADGARPAGHVPGGTQLPALTPAAGHREEAPGTPSLVHPSCLPGPRDEGRDHGTAAGRTGVTAREH SEQ ID COMMD3_Exon1_BLDAQADEAVLGFFIKQKCIEQRESRSLS NO: 263 MI1_Exon2 SEQ ID MED8_Exon7c_ELOKSASMHPYQRVLSQDGGCCELVPRGDEARRSPDPGLPSDGVPLAN NO: 264 VL1_Exon2DLHSPDLRVLRSLTWASHHG SEQ ID POLR2J3_Exon2_LODHTLGNIIKSRACFPFAFCRDCQFPEASPATLSVQPAEL NO: 265 C100134053_NA SEQ IDBGLAP_{circumflex over ( )}Exon2_PM QVQAQQQAWQVRSPAVQSPAKVQPLCPSRRAARNO: 266 F1_{circumflex over ( )}Exon5 SEQ ID TMEM199_Exon5_SQYIFTEMASRPRGAHWAGRDPEPGEGTRTRRAGAERGRHLGAHV NO: 267 ARM1_Exon2QAFGGDMPEAGGGRRPGRGAVLVPPHGPRAAAPLRAGAGQLRAARGPGGAATHGREARSRVALPARLLQGGRAASAARLPRSSGVGD SEQ ID C1QTNF6_Exon2_IL2PYINITILKGLPSSAPPCGCNGGPCSVLASAPPHPPPAPGYLLGICSGE NO: 268 RB_Exon2WHFPVHMLLQLESQHLLCLEPRWGSAGHFLPSPCLAGQTAVEPNL SEQ ID L0C100131434_NA_KLEEAGMLEMRPSTPCLHGAALHLHSGHGSGSRLTNSSCFPGTRRL NO: 269 FLJ44451_NALALQFTQQTGTVGHPTWQPVIR SEQ ID COX19_Exon2_CENTKEYLECRMERSRLGLLHSGRLHLPELLGNPPEYPPGQQGEVRPPGRL NO: 270 A1_Exon2GGGPSGVHGLPRERRRESQV SEQ ID ACSF2_Exon10_CHAINKINMKDLVRNLRKKLQHGKMDSKAPMSC NO: 271 D_{circumflex over ( )}Exon4SEQ ID TIMM23B_NA_LOC1 TMTGMLYKCTVSEMALDSPFCVLLSGS NO: 272 00132418_NASEQ ID NDUFA13_Exon4_YJ IIMKDVPDWKGLGAAAPTCRHGKSGA NO: 273 EFN3_Exon2SEQ ID ADHFE1_Exon13_C8 PALVKGTLPQYPVQPEEEPKALSTS NO: 274 orf46_NASEQ ID HPS4_Exon13_ASPH QLPALYEMTVSNSCTS NO: 275 D2_{circumflex over( )}Exon4 SEQ ID KIAA1267_Exon2_A DIYKQIRANKVSVWRQ NO: 276 RL17P1_Exon3SEQ ID LOC100129406_NA_ RTSALAERTHSIGHISTMLMAF NO: 277 CTTNBP2NL_NASEQ ID RNF216_Exon7_RBA LKGHYAITRKVYQPQSLHVSKSSRK NO: 278 K_Exon2 SEQ IDDEDD_Exon4_NIT1_ PYVTLKRRRAAPSGLGL NO: 279 Exon6 SEQ ID RAD54B_Exon3_LOCLATLDPPHTVQTWMRRHRLVPVHYR NO: 280 100128414_NA SEQ ID TOPORS_Exon2_DDPARPAPASSEKRCSIFRLRKTTRAQWRLPHFFSSSCWSSRRKAGSVA NO: 281 X58_Exon2 FWMPSEQ ID NDUFC2_Exon2_KCT LHPEDFPEEDVYCCGAERRG NO: 282 D14_Exon2 SEQ IDLRRC57_{circumflex over ( )}Exon5_SN CCPRLKILRLSALSVIRFICGF NO: 283AP23_Exon8 SEQ ID IPO11_NA_SLRN_NA LLSLLPSDNSLASKGP NO: 284 SEQ IDSNRPF_Exon2_CCDC VSVDGYMNMQQDFHLHLGNIETK NO: 285 38_{circumflex over( )}Exon12 SEQ ID NDUFB8_Exon4_SEC GDVYPVYQPVDRP NO: 286 31B_Exon2SEQ ID MIA_Exon3_RAB4B_ KPGKVDVKTDTSSSNSW NO: 287 Exon2 SEQ IDNIT1_Exon6_DEDD_ GSITGPAHWEQPVSS NO: 288 Exon4 SEQ ID Rnf139_Exon1_NdufIVLQIFLRLFETNTDTLLV NO: 289 b9_Exon2

As already noted, it is accepted by persons of skill in the art thathomologous sequences in closely related organisms that are accepted inthe art as suitable models one for another are expected to functionsimilarly. Thus, for example, the novopeptides disclosed in Table 6 maybe species-adapted for use in other closely related species such as, forexample, dog and mouse. A novopeptide identified with respect to a firstspecies may be species-adapted for use as a peptide component of ananti-cancer vaccine for administration in a second species in any manneroperable to better adapt the novopeptide to function in the secondspecies. By way of example, a novopeptide identified as having a firstdomain that aligns with all or part of a first gene of the firstspecies, and a second domain that aligns with all or part of a secondgene of the first species, may be adapted by modifying the first domainto improve its alignment with all or part of a gene of the secondspecies that is homologous to the first gene, and modifying the seconddomain to improve its alignment with all or part of a gene of the secondspecies that is homologous to the second gene. Accordingly, alsodisclosed herein are several of the novopeptides disclosed in Table 6,that have been species-adapted for dog (Table 7) and mouse (Table 8).

TABLE 7 RELATED SEQ ID SEQ. NOVOPEPTIDE SEQUENCE SEQ ID  THAP2_FDFCTHLKSMAIYSLFLLGVGCSLSSFLF NO: 242 Exon2_ TMEM1 9_Exon3 SEQ ID Rbm14_ GRVIECDVVKGAYQDGEAVHRKPAPGGHRA NO: 236 Exon1_ GDPLTLRAVWEGAGVRbm4b _Exon2 SEQ ID  IPO11- LLSLLPSDNSRLTSKGP NO: 306 SLRN SEQ ID  MIA-KPGKIDVKTDSNRTPTTQSAWSLDLG NO: 307 RAB4B SEQ ID  MED8- KSASMHPYQRVPNO: 308 ELOVL1 SEQ ID  ABHD14A- YTQEQFWAVKLTKRGA NO: 309 ACY1

TABLE 8 RELATED SEQ ID SEQ. NOVOPEPTIDE SEQUENCE SEQ ID  THAP2_FDFCTHIKSLVTFGLFLRGAGCSPSSFLL NO: 238 Exon2_ TMEM19_ Exon3 SEQ ID Rbm14_ GRVIECDVVKGSCQDGEAVHWKSAPGGHR NO: 232 Exon1_ AGDPLTLRAVREGAGMRbm4b_ Exon2 SEQ ID  Thap2 + FDFCTHIKSLGWWSDSS NO: 299 Tmem 19(2)SEQ ID  Rnf 139 + GLQIFLRLLGTSTGTLLA NO: 300 Ndufb 9 SEQ ID  Lats2 +IAPEVLLRKGTTFHGQ NO: 301 Xpo4 SEQ ID  Slc35a3 + QSVWIRNIQLASRNRLPNO: 302 Hiat 1 SEQ ID  Rbm14 + GRVIECDVVKGGMCVG NO: 303 Rbm4b SEQ ID Mia 1 + KPGKIDMKTDSNRTPTTLSAWSLDPGWST NO: 304 Rab4b(1) LGGRL SEQ ID Lats2 + KPGKIDMKTDTSSSNSW NO: 305 Xpo4

Example 17

From the candidate FS peptide sequences disclosed in Table 8 twocandidates were selected for in vivo confirmation. The candidates chosenwill be referred to herein as FS RBM (SEQ ID NO:232) and FS THAP2 (SEQID NO:238), these novopeptides having upstream in-frame portions thatalign with the mouse Rbm14 and Thap2 genes, respectively. Thesecandidates were chosen as the longest candidates the presence of whosesequence was confirmed by RT-PCR in cDNA of Tubo tumor, a mouse breastcancer cell line derived from the BALB-neuT mouse model intended to beused. The BALB-neuT mouse model is a transgenic mouse breast cancermodel, in which all ten mammary glands of a female mouse will developbreast tumor around 20 weeks of age, driven by overexpression of amutant rat Her2 gene.

FS RBM and FS THAP2 peptides were tested in vivo as candidate peptidecomponents of a prophylactic anti-cancer vaccine. BALB-neuT mice wereimmunized at 4 to 6 weeks old (primary genetic immunization); 8 to 10weeks old (boost genetic immunization) and 12 to 14 weeks old (boostpeptide immunization). All genetic immunization had the same geneticadjuvant for each bullet for gene gun immunization: 250 ng mixedLTA-LTB/pCMVi and 2.5 μgCpG 2216. All mice received two shots at eachear by gene gun for each genetic immunization. At primary immunization,the vaccine group got two shots of bullets containing 100 ng (highdosage) or 20 ng (low dosage) mixed FS antigen plasmids per bullet;while negative group got two shots of 100 ng mixed empty plasmids foreach shot. At boost genetic immunization, all mice received geneticimmunizations twice, three days apart, with dosage of 1 μg of mixed FSantigen plasmids or empty plasmids for each bullet. At boost peptideimmunization, each mouse received subcutaneously injected total 100 μlvaccine formulated with 50 μl Alum and 50 μgCpG 2216 as adjuvant; and 50μg total single FS peptide conjugated KLH or equally combined differentFS peptide conjugated KLH (vaccine group) or 50 μg KLH protein (negativegroup). There were 14 mice in the no treatment group; 30 mice in thenegative control group; 22 mice in the FS RBM group and 24 mice in theFS THAP2 group.

Both FS RBM and FS THAP2 showed significant prophylactic protection ininhibiting first tumor onset and tumor progression in BALB-neuT mice ascompared to the non-treated group and negative control group, as shownin FIGS. 13a and 13 b. The data shown combines two experiments withdifferent dosages at the primary genetic immunization (20 ng and 100 ngof the vector); no significant difference in results was noted asbetween the two dosages. FIG. 13a shows the percent tumor-free statusover the course of the experiment for the no-treatment group 1301, thenegative control 1303, the FS RBM group 1305, and the FS THAP2 group1307. FIG. 13b shows percent tumor-free status of the 10 mammary glandsover the course of the experiment for the no-treatment group 1301, thenegative control 1303, the FS RBM group 1305, and the FS THAP2 group1307. By both measures it can be seen that tumor progression issignificantly delayed for the immunized groups as compared to thecontrols.

Example 18

In vivo evaluation of prophylactic immunization using anti-cancervaccine compositions including several pooled FS antigens was performedwith results as shown in FIGS. 14a and 14b for four groups: controlgroup (44 mice, including no-treatment group and negative controlgroup); individual FS group (102 mice) including groups each immunizedwith one of FS SMC1A (SEQ ID NO:8), FS 1-78 (SEQ ID NO:2), FS RBM (SEQID NO: 232) or FS THAP2 (SEQ ID NO: 238); three antigen group, immunizedwith a vaccine composition wherein FS SMC1A, FS 1-78, and FS RBM werepooled; and four antigen group, immunized with a vaccine compositionwherein FS SMC1A, FS 1-78, FS THAP2, and FS RBM were pooled. Mice wereimmunized substantially in accordance with the protocol disclosed inExample 17 above.

FIG. 14a shows the percent tumor-free status over the course of theexperiment for the control group 1401, the individual FS group 1403, thethree antigen group 1407, and four antigen group 1405. FIG. 14b showspercent tumor-free status of the 10 mammary glands over the course ofthe experiment for the no-treatment group 1401, the individual FS group1403, the three antigen group 1407, and four antigen group 1405. It canbe seen that all the immunized groups showed significant protectionrelative to the control group, and the pooled three antigen group showedsuperior protection relative to the individual FS group.

Example 19

Sequences, 23 in number, for use as peptide components of an anti-cancervaccine were determined based on frameshifts as bioinformaticallypredicted to arise from microsatellite loci in mouse. These areenumerated in Table 9.

TABLE 9 Mouse SEQ ID NO Gene ID Sequence SEQ ID  NM_TLCMEVMLRWNTRELGYLYLQLCFLNTHFL NO: 310 153511.3HTSQEEKLLTLGRFLTWTSRCGSFVIRPL SEQ ID  NM_ ICMSPPLLWATLQAPETTSAACKASYRPEGNO: 311 053009.3 LYL SEQ ID  NM_ YFSCDKRCIKHYAGNKSLLTFSGY NO: 312010086.4 SEQ ID  NM_ KKSCPRYDPTLISLLYQCVS NO: 313 001114663.1 SEQ ID NM_ FLFPAFSCMPDLFITFLVTNTLLYFIQFSL NO: 314 146792.2 PC SEQ ID  NM_SAGTESDPSEEQICEAEGRPEGHFRGVLTY NO: 315 001081355.3 LPLL SEQ ID  NM_LSKTPSKKCSLRMNTKFYRSFTSLKSLIVT NO: 316 029998.3 FLRMVWWMLLRLEPISWKISEQ ID  NM_ HIQGQAEAGAVPGRALACWDLSAPVLPFTW NO: 317 001163189.1DEGVEIYRGPNTVVLL SEQ ID  NM_ FPPPGRCGLSSLDSHGL NO: 318 147003.1 SEQ ID NM_ HPQVCPPQGYLGQVMEQGQPHPLHPHSLQH NO: 319 009499.3RAPIVGVPGPQAWLLPLLEPNSGK SEQ ID  NM_ KKRASPLLGRTPLATRIRETLAHHLCYQKNO: 320 130448.3 SEQ ID  NM_ IPSWGRSFYCGNVLPSYHSEWWQL NO: 321 033618.3SEQ ID  NM_ GLFHARISVQEQYQGELPLLGGKCGERSL NO: 322 001081302.1 SEQ ID NM_ GVNGARRNSRIGEFRKVTIFLTARV NO: 323 028664.1 SEQ ID  NM_VTAGYQEEEMEASACGAKGPGLAPWPPSW NO: 324 001034881.3LALQDSLLCVVVALADLRRKSCCS SEQ ID  NM_ LSLSFLHRWMDKTVGTI NO: 325001033226.4 SEQ ID  NM_ TLCMEVMLRWNTRELGYLYLQLCFLNTHFL NO: 326 153511.3HTSQEEKLLTLGRFLTWTSRCGSFVIRPL SEQ ID  NM_ RVYSKLENQKAAKEGGNTQVKRKGGHRASANO: 327 001160399.1 FSKQSRR SEQ ID  NM_ AKEQAAAEAAEEQAAACRCGSQPVSLCQCQNO: 328 025441.3 KIL SEQ ID  NM_ RQKKIRPPKKKRSIQGQRQKPPRDHRCECD NO: 329001109759.1 QLFCFFWWGGNP SEQ ID  NM_ LLCVVFGKFVIPRSTFRHTGCHSEYFVFNFNO: 330 028787.4 WTFYFNPCSCISE SEQ ID  NM_ LKSAPLQATTTLKLIPVMRGTATELNO: 331 027009.2 SEQ ID  NM_ VLPNLPSQSSTF NO: 332 001081345.2

The first three peptides (SEQ ID NO: 310, SEQ ID NO: 311, and SEQ ID NO:312) were tested in vivo and shown effective in prophylacticallyinhibiting tumor progression in BALB/C-4T1 mouse transplant breast tumormodel. Primary genetic immunization was administered with the threepeptide components (10 mice) or with empty vector (negative controls, 4mice) at 7 weeks old and 13 weeks old. Three weeks after the lastimmunization, all mice were challenged with 5,000 4T1 tumor cells. Tumorsize was measured each day after palpable tumors appeared. FIG. 15 showsaverage tumor size for the negative control group 1501 and the immunizedgroup 1503. Error bars show standard error.

Human and dog peptide candidate sequences homologous to those of Table 9are shown in Tables 10 and 11, respectively (not all of the mousesequences of Table 9 have homologs in human and dog).

TABLE 10 SEQ ID Human NO Gene ID Sequence SEQ ID  NM_SASTESNSSEKQICKAEGRLEKCL NO: 333 015866.4 SEQ ID  NM_QSRIPSKKCSLRKSTKSYKSCTNLRNLAPT NO: 334 152512.3 SEQ ID  NM_PPHPQVCPLRGSQLQRTEQGEDHPLHPLSR NO: 335 003370.3QHRALVVGELGPQAWPQLLLEPNSGKSASR RRPQGGPQPPKLRVVEAEVGDSWKR SEQ ID  NM_IPCWGRPFYCGNVLPSYHSEWWQL NO: 336 007192.3 SEQ ID  NM_ KEGLLHAGIPVNO: 337 007118.2 SEQ ID  NM_ RVNSDLEN NO: 338 152549.2 SEQ ID  NM_LLCVVFGKFVISRSTFRHTSCYS NO: 339 025181.3 SEQ ID  NM_LKLAPLQVTTTLKLILVMLEIVTE NO: 340 002915.3 SEQ ID  NM_ KLLNLSCQSCTNO: 341 001271.3

TABLE 11 SEQ ID Dog Gene NO ID Sequence SEQ ID  XM_QSRIPSKKCSLRKSTKSYKSCTSLKSLVPT NO: 342 850372.3 SEQ ID  NM_HPLVCPSQGALRQGTEQGEAHPLRPLSPQH NO: 343 001003256.1KAPVVGEQGPPVLQQPLPEPNSGKSASKRR PQGGPQSLKQRALEARAGG SEQ ID  XM_VPGRGRPFYCGNVLPSYHSEWWQL NO: 344 846473.3 SEQ ID  XM_ KKGLLDARIPVNO: 345 005639663.1 SEQ ID  XM_ RVYSELENQKAAKEGGNFQVKGKSREPIST NO: 346005626355.1 LS SEQ ID  XM_ LLCVVSGKFVVSRSTFRHTGCYS NO: 347 533330.4SEQ ID  XM_ ILLNLSSESCTF NO: 348 005618324.1

Example 20

Sequences, 142 in number, for use as peptide components of ananti-cancer vaccine were determined by comparing NCBI EST databasesequences with human RefSeq generally according to the criteriadescribed in Example 16 above and identifying sequences wherein theupstream non-frameshifted domain and the conjoined frameshifted domaincorresponded to different genes. These are tabulated in Table 12.Sequences shown are the 10 residues of the upstream non-frameshifteddomain immediately adjacent to the junction point, together with thefull sequence of the frameshifted domain up to the first stop codon.

TABLE 12 SEQ ID NO Upstream ID Downstream ID Sequence SEQ ID NO: 349NM001080414.2 NM198530.2 MDVTVSELLEN SEQ ID NO: 350 NM001127716.1NM004589.2 MFRAAAPGQLS SEQ ID NO: 351 NM001005409.1 NM001127398.1MPAGPVQAVPS SEQ ID NO: 352 NM182568.3 NM002767.2 MASWGGEKRGLSEQ ID NO: 353 NM000094.3 NM033199.3 MTLRLLVAALA SEQ ID NO: 354NM004536.2 NM002538.2 MATQQKASDEG SEQ ID NO: 355 XM001725801.1XM377073.4 MTPVRMQHSLR SEQ ID NO: 356 XM001725801.1 NM182905.3MTPVRMQHSLR SEQ ID NO: 357 XM001717526.1 NM018081.1 MGSKIYSYEFVSEQ ID NO: 358 NM001131028.1 XM001715307.1 MEEDEFIGEKV SEQ ID NO: 359NM080664.2 NM016106.2 MAEGSRIPQAGF SEQ ID NO: 360 NM016616.3 NM017549.3MASKKREVQLII SEQ ID NO: 361 NM004136.2 NM181354.4 MDAPKAGYAFEISEQ ID NO: 362 NM015004.3 NM003278.2 MASVTLSEAESA SEQ ID NO: 363NM013293.3 NM024322.1 MSDVEENNFEVF SEQ ID NO: 364 NM005085.2 NM175878.3MGDEMDAMIPAP SEQ ID NO: 365 XM001129743.1 NM001031716.1 MAFGEVEHTDDASEQ ID NO: 366 XM001721832.1 NM053052.2 MRARLRFLPSDC SEQ ID NO: 367NM001145.3 NM002937.3 MVMGLGVLLLHL SEQ ID NO: 368 NM033542.2NM001048225.1 MAGQFRSYVWWS SEQ ID NO: 369 NM018281.2 NM004462.3MLRVLCLLRPDLP SEQ ID NO: 370 NM002300.5 NM016078.4 MATLKEKLIAGSFSEQ ID NO: 371 NM022780.2 NM022662.2 MDQCVTVEREDSL SEQ ID NO: 372XM001717653.1 NM152705.1 MYFLNDALCAGKQ SEQ ID NO: 373 XM001719888.1NM004906.3 MAERGGWREADSR SEQ ID NO: 374 XM001717673.1 NM201554.1MGGGPAREKGPTL SEQ ID NO: 375 NM005004.2 NM015490.3 MAVARAGVLGDRPSEQ ID NO: 376 NM020984.3 NM001042427.1 MAAKTPSSEELCWP SEQ ID NO: 377NM016406.3 NM005614.3 MADEATRRVVGNPH SEQ ID NO: 378 NM032775.2NM182895.1 MAEEQEFTQLRDQV SEQ ID NO: 379 NM030940.3 NM016548.2MSASLVRATVDSQR SEQ ID NO: 380 NM001002001.1 XM001715885.1 MPHIDNDVKLGADKSEQ ID NO: 381 NM001031827.1 NM015092.3 MASAKSLDRWFCTE SEQ ID NO: 382XM001718309.1 NM015681.3 MRTGSRAPSEVSRV SEQ ID NO: 383 XM943571.2NM022483.3 MRRPRLPAQAVLEM SEQ ID NO: 384 XM001716831.1 NM017897.1MYFHKKPPALGMCF SEQ ID NO: 385 NM001013845.1 XM001720996.1 MKFGCLSFRQLQNPSEQ ID NO: 386 NM178124.3 XM001720996.1 MKFGCLSFRQLQNP SEQ ID NO: 387NM014251.2 NM006304.1 MAAAKVALTKLGWLR SEQ ID NO: 388 NM001928.2NM005481.2 MHSWERLAVLTLTRS SEQ ID NO: 389 NM016374.5 NM001037277.1MKISFEV SEQ ID NO: 390 NM005600.1 NM032998.2 MLGFITRPPHQPVSSSEQ ID NO: 391 NM001487.2 NM002905.3 MLSRLLKEHQGFLVW SEQ ID NO: 392NM001032363.1 NM182744.2 MSESELGRKWLWRPRA SEQ ID NO: 393 NM001010974.1NM001099432.1 MQRTGGGAPRVPLTGA SEQ ID NO: 394 NM006868.3 NM001402.5MMAIRELKVCCRENYP SEQ ID NO: 395 NM015215.1 NM018948.2 MWRAEGKWLPGMKATGSEQ ID NO: 396 NM001002840.1 NM001013739.2 MQHREVRVKCNTEKSASEQ ID NO: 397 XM001130925.2 XM001126402.2 MASASCSPGRWNNPKFSEQ ID NO: 398 NM001134779.1 NM181506.4 MVQPIIHLGYLASKGP SEQ ID NO: 399NM006328.3 NM002896.2 MKIFVGNVDGGGMCVG SEQ ID NO: 400 NM152350.2NM014288.3 MFPGSLSRGRHDVAIKS SEQ ID NO: 401 NM015004.3 NM003278.2MASVTLSEAECCEHKDV SEQ ID NO: 402 NM014662.2 NM004147.3 MRTTKVYKLVLWSGVSLSEQ ID NO: 403 NM006837.2 NM012207.2 MAASGSGMAQVTMGKAA SEQ ID NO: 404NM001113756.1 NM005138.2 MAALMTPGTGASDPCCC SEQ ID NO: 405 NM032998.2NM005600.1 MAGLKRRASQAPSGLGL SEQ ID NO: 406 NM001113756.1 NM005138.2MAALMTPGTGASDPCCC SEQ ID NO: 407 XM001714553.1 NM001114620.1MWNAALPGPTHGCLLIP SEQ ID NO: 408 NM133638.3 NM014031.3MRLTHICCCCERRKCRNH SEQ ID NO: 409 NM001005354.2 NM001128914.1MWNPNAGGPPFLAFTPNQ SEQ ID NO: 410 XM001715546.1 NM020987.2MANETLFSSPSLMPMQVT SEQ ID NO: 411 NM004388.2 NM005274.2MSRPQLRRWRKFPRQLQT SEQ ID NO: 412 NM016622.3 NM022912.2MAASAFAGAVSRGFGGRG SEQ ID NO: 413 NM000296.2 XM001725512.1MPPAAPARLAVDGRMATWM SEQ ID NO: 414 NM001098504.1 NM005839.3LPTGFVAPILKSLGLKMML SEQ ID NO: 415 NM012112.4 NM001031711.2MSQVKSSYSYMEMCGMHMS SEQ ID NO: 416 NM018233.3 NM031885.2MNGKRPAEPGPRKKLSPLA SEQ ID NO: 417 NM022739.3 XM001719702.1MSNPGGRRNGVLSRVMTTP SEQ ID NO: 418 NM182471.1 NM001005.3MSKPHSEAGTGSANIQEEEVCR SEQ ID NO: 419 XM001722372.1 NM018704.2MAGRPGSQEQSIGHISTMLMAF SEQ ID NO: 420 NM153260.2 NM003825.2MGNSALRAHVSALSVIRFICGF SEQ ID NO: 421 NM014774.2 NM022745.3MKKRKELNALRCLGETVCEQRIH SEQ ID NO: 422 NM016040.3 NM015361.2MGDKIWLPFPIKLYPTSSKTTKE SEQ ID NO: 423 XM001714221.1 XM001714962.1MRTLPLRFAGYFNSRPHLCPAGS SEQ ID NO: 424 NM182533.2 XM001714962.1MEAARRPRLGYFNSRPHLCPAGS SEQ ID NO: 425 NM003095.2 NM182496.1MSLPLNPKPFQDFHLHLGNIETK SEQ ID NO: 426 NM001127184.1 NM015147.2MSAEVIHQVEDILLSAGRADLLAV SEQ ID NO: 427 NM030980.1 NM052998.2MSTLLLNLDFGSAAKAADGCRTGG SEQ ID NO: 428 NM001630.2 XM001715226.1MAWWKAWDSGILPLRNALLLG SEQ ID NO: 429 NM207116.1 NM021163.3MEEGNNNEEVVYQPQSLHVSKSSRK SEQ ID NO: 430 NM012415.2 XM001722896.1MRRSAAPSQLQTWMRRHRLVPVHYR SEQ ID NO: 431 NM182641.3 NM002266.2MRGRRGRPPKSFLPLSHNHVHQREC SEQ ID NO: 432 NM015965.5 NM198537.2MQEPRRVTPCGLGAAAPTCRHGKSGA SEQ ID NO: 433 NM005833.2 XM001715611.1MKQLPVLEPGTGFHRVSQDGLDLLTS SEQ ID NO: 434 NM001128208.1 NM001100916.1MAGIKALISLRVQMSASGTYPSSPGL SEQ ID NO: 435 NM014637.2 XM001720291.1MLGWIKRLIRSLPAGFIQPHVSKHCLG SEQ ID NO: 436 XM928114.3 XM001719607.1MEGGGGSGNKVSEMALDSPFCVLLSGS SEQ ID NO: 437 NM016026.3 NM006370.1MVELMFPLLLWQRWRRSYVMHPCLSETP SEQ ID NO: 438 NM022730.1 NM002867.2MAGEQKPSSNVETGFHLVSQDGLDLLTS SEQ ID NO: 439 NM006454.2 NM006503.2MELNSLLILLSRQGGSEDPAGAAESDGWI SEQ ID NO: 440 NM025149.4 NM001267.2MAVYVGMLRLRNLRKKLQHGKMDSKAPMSC SEQ ID NO: 441 NM000031.5 NM024939.2MQPQSVLHSGSIVFEARGDKAEIRDGALQQG SEQ ID NO: 442 NM006116.2 NM018133.2MAAQRRSLLQTFATRSYCTHQLHLPTLCLQNL SEQ ID NO: 443 NM007221.2 NM199173.3MAEASSANLGVRSPAVQSPAKVQPLCPSRRAAR SEQ ID NO: 444 NM007221.2 NM199173.3MAEASSANLGVRSPAVQSPAKVQPLCPSRRAAR SEQ ID NO: 445 NM007221.2 NM199173.3MAEASSANLGVRSPAVQSPAKVQPLCPSRRAAR SEQ ID NO: 446 NM005358.4 NM000358.2MKKIRICHIFKCWTPWSAMSTLSCSMPSATIWW AGES SEQ ID NO: 447 NM016075.2NM018676.2 MDRFVWTSGLCFWGKKSQSGREKGHEETKDLGF CQSE SEQ ID NO: 448NM001012754.2 NM170719.2 MARFWVCVAGFQGGLQSSSCYDIFLWNNPRKSI SQRKTSEQ ID NO: 449 NM014637.2 XM001720291.1MLGWIKRLIRVPSSWLYSAPRVQALSGIALYWKT WPIL SEQ ID NO: 450 NM018049.1NM032482.2 MRYNEKELQATETPRDPRQACPASQGMLRAMD SREIQK SEQ ID NO: 451NM145301.1 NM173622.3 MLQQDSNDDTEPSEMSCTRNFKREFSAGRRGRQ DIRTRISEQ ID NO: 452 NM024061.3 XM001716989.1MEEIPAQEAADFQFPSLMGSPSWNRIYRSLIWKL RLEKS SEQ ID NO: 453 XM001715735.1NM014393.1 MVARPLHSTERLPTTRTLAYFLKIEEKKEDRKRRTQ RYTK SEQ ID NO: 454XM001719043.1 NM138401.2 MSVSVLAPAGSPAPSRGHPPALARASRRNLATSC ALVLWAASEQ ID NO: 455 NM006098.4 NM015458.3 MTEQMTLRGTVRPGWSAVVRSRLTASSASRVHTILLPQPPE SEQ ID NO: 456 NM001013839.1 XM001725398.1MIPPQEASARISPGHEHDFRVKHLSEALNDKHGPL AGEYRSPA SEQ ID NO: 457 NM018696.2NM005359.5 MSMDVTFLGTYPEYMSNNFPCNVSCCFSLFPKD QNCFRNWRHI SEQ ID NO: 458NM001034.1 NM182626.1 MLSLRVPLAPLGDREVQSRWSPGPRGDSTPVRE METNHPPSVRGSEQ ID NO: 459 XM001714450.1 XM001714789.1MGSSAVQSQLWHFSTPLEPMPRRNKGCAASPW LTQWPRPRKSQR SEQ ID NO: 460XM001721832.1 NM023007.1 MRARLRFLPSTGLRCSLLCLDRPGRARPHLHHTQC EEGLGTHHSSEQ ID NO: 461 NM006328.3 NM002896.2 MKIFVGNVDGGSCQDGEAVHRKPAPGGYRAGDSLTLRAVWEGAGM SEQ ID NO: 462 NM001006947.1 NM017771.3MAGIIKKQILNILFECKEEFLWKTAGRLLEDTVTLIC LTTAYRLQA SEQ ID NO: 463NM001031665.1 NM001105552.1 MLREEATKKSGSIDIQGCGHRILSGGVEMPGPCSEDSIQRCDVGEL SEQ ID NO: 464 NM003002.1 NM031275.4MAVLWRLSAVCLQCQIVHSCPLLENQIHLSLKFPD YFIKMKPWRKI SEQ ID NO: 465NM006743.3 XM001127723.2 MSSEEGKLFVLGGGGQRQTEPGRLGGDDWSCM RPRSHLWMVDLPWASEQ ID NO: 466 NM207116.1 NM018053.2 MEEGNNNEEVGLASAHPSWASRGHCSTTTGPCAPASPPSRSWAWAPP SEQ ID NO: 467 NM001079673.1 NM001984.1MAEHPPLLDTNRLPFGASKKQSAIGQEKNGIEAD FQQQVLWGIAESF SEQ ID NO: 468NM005802.2 NM014314.3 MGSQPPLGSPKRCSIFRLRKTTRAQWRLPHFFSSSCWSSRRKAGSVAFWMP SEQ ID NO: 469 NM007273.3 NM181642.2MAQNLKDLAGALRGPARHRTLQGEHPALVLQPL QRTLRPLYLWWLLRQQEQL SEQ ID NO: 470NM001320.5 NM021221.2 MSSSEEVSWISSCSRHPDVPLLPRRRPFCRMHFRLREVYHQQLIPVHGDHHLL SEQ ID NO: 471 XM001132260.1 NM018427.3MAKRRRPKKRDSQRHFWNISGKNCRTQVILPSSG RLLEIILEAFWQELNLFLLLL SEQ ID NO: 472NM000972.2 NM000787.3 MPKGKKAKGKLSHPPGPGGVPGALMECQLHPGGHPFPAPGAEAQGWRPVWDVRPWRA SEQ ID NO: 473 XM001723697.1 XM001716892.1MRGQTEEVVASASWAVPSTCCFRSCKPLLAMAS GYLWSRQEQLERRWILPRVAMPSASSGFSEQ ID NO: 474 NM007221.2 NM199173.3 MAEASSANLGCEAQRCRVQQRCSLCVQAGGQRGSEETQALPVSMAGSPSPLPGSPGAQEGGV SEQ ID NO: 475 XM001721832.1 NM053052.2MRARLRFLPSEAEEAWTLAHTDPGTDEQGCLHP HLAVHLLPGAQEAMGYWTAVLNIAVAQVHDSEQ ID NO: 476 NM006396.1 NM001098785.1 MALNGAEVDDGGAAAVGHVLVVPAVGPVRVNPGLQTPVPRPELLPGPVILPPFGQLLPTGCGPV SEQ ID NO: 477 NM153260.2 NM130798.1MGNSALRAHVMLLHGQTQRPQHLLSSVKVFSVS LMSLFIWSKPSSMRFSCSFCSSSIVMVLIPASSEQ ID NO: 478 XM001713865.1 XM001714058.1MDPASRGCLGRPSTPCLHGAALHLHSGHGSGSRL TNSSCFPGTRRLLALQFTQQTGTVGHPTWQPVIRSEQ ID NO: 479 NM000985.3 NM001035005.2MVRYSLDPENLRSSSLGKWCAFLVSSFQFCSGSTK NSWSHIYTLWFPPSLVVYGLRKQYKNPMIQTKAKSEQ ID NO: 480 NM017882.1 NM033429.2 MEATRRRQHLRKWRAGFLHFSDHYAHANKTRRPKERNSSSHVDGGQGEERLRHGVRPAVKTHESGG EAHPQGSG SEQ ID NO: 481 NM012447.2NM005395.2 MSSPLQRAVGLDYVDMEIHLPLSTAPLPAPLPSPPLHDDVWLGDNHTPQKLDGCSSPTSHPRMLSSHQ GPVAATPPG SEQ ID NO: 482 NM015001.2NM001042704.1 MVRETRHLWVFWNPGAEDHVGGCDLGGLPGYQELWGQGGGPLLHPGHRQHPVPGQSGPFRAPV CNDRCLPGPCAHHDHRGA SEQ ID NO: 483NM003755.3 NM207346.2 MPTGDFDSKPPWSFSISLCCRQHTFLMEVPGCWRSLGAWKSSLMFTRPTLWPHSERITLANPMPGCA LVDLMSLSQTSAASSGCLTRVGMSLSEQ ID NO: 484 NM182486.1 NM000878.2 MQWLRVRESPLPSSAPPCGCNGGPCSVLASAPPHPPPAPGYLLGICSGEWHFPVHMLLQLESQHLLCL EPRWGSAGHFLPSPCLAGQTAVEPNLSEQ ID NO: 485 NM020987.2 NM005888.2 MAHAASQLKKSGCVIAILGKRCSRPWRTWRGRTPSTRHICSWCTMVSGTSAAAPQGPRASPAALAT WQPPPWKSSIAVTMDLADSLSFVDLEELLAVAQHIQHWFL SEQ ID NO: 486 NM000921.3 NM001128220.1MAVPGDAARVEQDCQWRLPQRPPGAEPQPHR ARCGPHHEPARQPPVHLPPQPWPGAAPRTDQEQPGRQQASRPLAHTDPGGAAAEPALRDHLCLGW RPQLQPLALPHRAQRLCRARACQLPEATERCHHLSEQ ID NO: 487 NM178276.4 XM001722866.1MSAQCCAGQLATKVPTSRASSAGAGPSSGSRWP PAGYACCCTCVRWSLPSAAPPGSSLCDDIGGPLGFVGLQPGKCHLLNSVPGAGTGALCLSGSEKALRLHS GTSSLLFPPSYFGKFSSRSSHVFSIQLSCHSFSSEQ ID NO: 488 NM004083.4 NM004990.2MAAESLPFSFLPLGASGGFPSATANCFFRSKSFATSAATSFLSAFCAFSSRTMFPCFVTSSISACICCGLAVV TVSTTAGFGDVFAWPPPKRCLKLSIWSFSNFWNKGLTVPIWCPAGKVHRKFVSRILQAGGGSCSWAWI VALTVGM SEQ ID NO: 489 XM001716912.1NM199511.1 MREVQKDQRSPFSTSHSCVFPQPGKHINPSAFTPGNKETKPDYGGKGDKKDPGTLKRERVRFRNRQD WTLRDVLCQHKGLAHTDTRDRGETADKWRYKDLEGQLLSPHPTCPEGKKTQPERKGIGSALRSGQAVD LWRYSESWSCSTPLLYCSPSVKRERAGRKSSSSRKLSWEPNF SEQ ID NO: 490 NM006871.3 NM139247.2MSCVKLWPSGADLRPELPDHCAVRAGRLLAAAG PRFPGAATAALDASPVRLGMGRAASARPRLPVHRGRGERLGPGVLFSLRHLHGVCHAALGHAGRRRR GPRLLTLASAGPRAVSWATAGLTACTAAAVGSKRSAVPVRERGRSVPQGADGARPAGHVPGGTQLPA LTPAAGHREEAPGTPSLVHPSCLPGPRDEGRDHGTAAGRTGVTAREH

Example 21

Sequences tabulated in Table 13 for use as peptide components of ananti-cancer vaccine were determined by identifying tumor-associatedframeshift alternative splicing variant peptides. The NCBI EST sequencedatabase was screened against the human RefSeq database to identifysingle EST sequences that align with two RefSeqs, in which an exon isskipped, and in which the sequence downstream of the splice isframeshifted relative to the normal wild type when the EST sequence istranslated. From these, sequences were identified wherein either thesequence corresponded to at least 3 tumor EST sequences and nonon-cancerous EST sequences, or in which the sequence corresponded to atleast three times as many tumor EST sequences as non-cancerous ESTsequences.

TABLE 13 SEQ ID NO Name ID Sequence SEQ ID NO: 491 C11orf2 NM0132652PCTGLSLHPMAPRIWSRWSFPAGRCQDRPNKHVWPPQ KKKKKKKKKKK SEQ ID NO: 492C20orf96 NM080571.1 CFTSSPLRW SEQ ID NO: 493 CYBASC3 NM001161452.1LLLQLRPGSRPFPVTYVSVTGRQPYKSW SEQ ID NO: 494 KRT8 NM0022733LLRSRHSTRILPTAAGLRLRACTRSSMRSCRAWLGSTGM TCGAQRLRSLR SEQ ID NO: 495 MVKNM001114185.1 GGPRRIWS SEQ ID NO: 496 NAA10 NM003491.2RSVKWSPNTMQMGRTPMP SEQ ID NO: 497 PDCD2 NM001199462.1GLWLFRPQNVLQMPQSILLQQGASDPRLEIGT SEQ ID NO: 498 RPS3A NM001006.3FGKAHGASW SEQ ID NO: 499 TFE3 NM006521.4 CSAQARNRSEDETQPLPLGTLLAFSEQ ID NO: 500 HNRNPA2B NM031243.2 KEGVLLQVTNEEVVNHRVFKK SEQ ID NO: 501NOLI2 NM0243112 VPTACCRCCFCWDV SEQ ID NO: 502 RPLP0 NM001002.3GVRQWQHLQP SEQ ID NO: 503 DPH2 NM001384.4LPCSSLTSYWEMLWLWLHDWRRRQGQRCSFWVTQPT AAAAWMCWVLSKLELRLSYILALPASEQ ID NO: 504 GNB2LI NM006098.4 GWPGHVMGSQRRQTPLHARWWGHHQRPVLQPSEQ ID NO: 505 RPL8 NM000973.3 IRELCHRYLPQP SEQ ID NO: 506 IGFLR1NM024660.2 NCPVWRHNPCLASWMSWRCWKS SEQ ID NO: 507 KARS NM001130089.1VGSMPKELLGESSSSMIFEERG SEQ ID NO: 508 MRPS28 NM014018.2 EIPERNQGPVAAIRSSEQ ID NO: 509 HNRNPA2B NM031243.2 EGVLLQVTNEEVVNHRVFKK SEQ ID NO: 510SMC1A NM006306.2 CCGIYCHEEPQREDSSI SEQ ID NO: 511 NRM NM007243.1AGDAVLGAHTQRPCVVGGSG SEQ ID NO: 512 PRSS27 NM031948.3 PLRRPCTRSCWGQGSSEQ ID NO: 513 TXN2 NM012473.3 CQRCPLCWP SEQ ID NO: 514 RDH11 NM016026.3SLPPNPSAARETKGISPIKDSKCVFPRTSPGKDPLP SEQ ID NO: 515 BORA NM024808.2FSLKMSSYPLLGLIMKGNSFHNVIPVNALT SEQ ID NO: 516 RPS3 NM001005.3GLLWCAAVHHGEWGQRLRGCGVWETPRTEG SEQ ID NO: 517 SAALI NM138421.2GDGGSGSKGRPVEQTEVFLCISKPSSFL SEQ ID NO: 518 SEMA3B NM00I0059141LPQQDLWHLQFHQGLPRRCHPVCAEPPPHVQLCPAHW GAPSFPTSWSQLHLHSNCRGPGCSRSEQ ID NO: 519 FPGS NM0010180781 AGPSPGTWTVRTPPAARRPACAGSARRCRAARGRAVAPRFESCSSMLPGTGTRRPC SEQ ID NO: 520 SLC13A3 NM001193342.1GIGAVCMDWWAAAPPGECAPRPGCAAHHCGHRLLH SEQ ID NO: 521 ARHGEFI NM199002.1GVGGGILPPETPPVSAWGELCPPAWLHL SEQ ID NO: 522 FANCI NM001113378.1VSPGVSELRRNSKKYGKAGEAVWFSSDPPVLFFHFLRTE SEQ ID NO: 523 SARS2 NM017827.3LHARAPGPRGPPLLCPCCLRVSH SEQ ID NO: 524 CAPN3 NM000070.2 CLQKHLPVALSTSLCSEQ ID NO: 525 SPAG5 NM006461.3 ISVSIMWTQRRKL SEQ ID NO: 526 ZNF263NM005741.4 SHSQSGGPRHPGGTRRKAMGSQCPELQGGPEPQRPSS RRREI SEQ ID NO: 527DFFA NM213566.1 SPKLPLVRRWMQ SEQ ID NO: 528 NSLI NM001042549.1GAKPGGLALGAV SEQ ID NO: 529 C17orf85 NM018553.3CYQHPFPKKSQFPGAYWTSFEGEEEGSGQLTLPGP SEQ ID NO: 530 CIRHIA NM032830.2LLSSHHPLKRRNLEP SEQ ID NO: 531 APEH NM001640.3 SPSQAMWATRMSEQ ID NO: 532 DPP3 NM130443.2 HFPACQLLPLCDLISSALPYVE SEQ ID NO: 533EEF1AI NM0014025 CLQNWWYWYCSCWPSGDWCSQTRYGGHLCSSQRYN GSKICRNAPSEQ ID NO: 534 ARMC8 NM014154.2 RHEKCCNWKQQAESQSHCFRSCSKIVVLASARNLKHRAEN SEQ ID NO: 535 TMEM179 NM207379.1QFRTPGWPLKALAGRGWPEDASPGQEPSKGAGRGWA SEQ ID NO: 536 VASP NM003370.3WPQLLLEPNSGKSASRRRPQGGPQPPKLRVVEAEVGDS WKR SEQ ID NO: 537 MRPL43NM032112.2 PASGGSDLVNHSFLCKWHP SEQ ID NO: 538 MRPL43 NM032112.2CLLLGAVTL SEQ ID NO: 539 DEDD NM001039712.1 AAAAAHHHSPRPAALRHPQEETGCVPSEQ ID NO: 540 AURKB NM004217.2 DHGGVGRCSNVLPWEEGDSQRHKARKSALRAQGRAED CSEQ ID NO: 541 CRCP NM001040648.1 TSASQIQAILVP SEQ ID NO: 542 WTAPNM152858.1 GLMASDYSEEVATSEKFPF SEQ ID NO: 543 NUP43 NM198887.1QENCSNPGGRGCSDPRSCHFTPAWAKEQNAISKNIHI SEQ ID NO: 544 SRSF5 NM006925.3VKGVLHSLTAAGQTH SEQ ID NO: 545 HSPHI NM006644.2 DSCGIVNSY SEQ ID NO: 546IGFLRI NM024660.2 NCPVWRHNPCLASWMSWRCWKSDEVFALPLAHLLQTQNQGYTHFCRGGHFRYTLPVFLHGPHRVWG SEQ ID NO: 547 NUDT8 NM1818431LTAVITEFALQLLAPGTYQPRLAGLTCSGAEGLARPKQPL ASPCQASSTPGLNKGL SEQ ID NO: 548STK25 NM006374.3 KHQAMDHHGVPGRRLSTGLA SEQ ID NO: 549 TNFAIP2 NM0062912PRAAVSGIQQWWNGRQNWKRKKEKMSSRLAGAFRVL WRAVSTASIRRHIQVAPRPLQAGPAMGPSEQ ID NO: 550 TTLLI2 NM015140.3 LIVGGGAPDRKGFQ SEQ ID NO: 551 UQCCNM001184977.1 GVRCLIHSIHGFL SEQ ID NO: 552 WDR34 NM0528443VAARAWAQPPLPGAECGHRREGATLAGHRGRPAAAHRGLRPGHAAAATEHQAQEASPRGDRGGRHGSGLLQLHRDSRGSGRNGRHPEREGDHAKPERPPGLLPGQSEEPGDR EPEAG SEQ ID NO: 553 KRT18NM1991871 EQNPGALGEEGTPGQRLEPLLQDHRGPEGSDLRKYCGQ CPHRSAD SEQ ID NO: 554NOP16 NM016391.4 SGKTSSILCRRGRWRWS SEQ ID NO: 555 SNX27 NM030918.5HFPDGEVTAERCGHLAFPYPLPFPSPPSSYSFHVPFQTE SEQ ID NO: 556 RAB25 NM020387.2GTIVVQWGPSWCLT SEQ ID NO: 557 ATP5B NM001686.3 TTNPSRISLPSWVWMNFLRKTSSEQ ID NO: 558 BFAR NM0165612 WSCSSITGAAGNLNTTSWSTRLWPNGRRKKLSSGWSSWALGHLFTGKGFYLNE SEQ ID NO: 559 C160rf62 NM020314.5 GSADRDDGKVSEQ ID NO: 560 C190rf40 NM152266.3DAAFFMSPKLrWWQEMATERGLFGLEIPIILKELRVQGTLVHCPTRHLSQRRGPGRQRGNSLPEPSSMLTCPQQPHR AT SEQ ID NO: 561 C90rfi40NM1784483 FPAAPGLQGCPRTGPSQPSMQLPSYPEDGSGLSRGHKDVRPGPPGQERVQVLRACAPQPQHQVDCSAVGGPVAAREKPPVSRLGSAHQGLPTSAFEGACHALGDPGIFTGLEAG DRTVSVPG SEQ ID NO: 562 DERANM015954.2 LLQPPFVFIPPGCVML SEQ ID NO: 563 EXOSC2 NM014285.5 GFWSRFPPPWSEQ ID NO: 564 GTPBP5 NM015666.3 GPRGHAGEGGRQSCGRPVLRGR SEQ ID NO: 565HSPH1 NM006644.2 DSCGIVNSY SEQ ID NO: 566 IST1 NM0147612IVGPGPKPEASAKLPSRPADNYDNFVLPELPSVPDTLPTA SAGASTSASEDIDFDDLSRRFEELSEQ ID NO: 567 MAGED2 NM1774331 RCQPDRHSHIWALRWPWWSWCQHQWQLWCLWFLL QVSEQ ID NO: 568 MED19 NM153450.1 ETPSDSDHKKKKKKKEEDPERKRKKKEKKKKKVESEQ ID NO: 569 MRPL2 NM015950.3 AGNVRSNSRPSIQR SEQ ID NO: 570 MTFR1NM001145839.1 LHWGSTKVHLLLI SEQ ID NO: 571 MVK NM0004312 GGPRRIWSSEQ ID NO: 572 NUPL2 NM007342.2 AKFCPTFNKSMEEQGK SEQ ID NO: 573 PEX13NM002618.3 DYRRLPPGPANFFCIFSRDGVSPCYPGWSPSPDLVMSPL RSPKVLGLQASEQ ID NO: 574 PSPH NM004577.3 CDLNSLCIFVAIFHTKCFKCGESIKHLYSGLWMVVRSVWIMQASLLGEPEEVALGPMGVVAATLEVVGTRAM SEQ ID NO: 575 RBM3 NM006743.4GVAGIMTVDLEGMDMDMDVPETIMAETRVVMTATQE EITETIMTT SEQ ID NO: 576 RNF217NM1525532 GLFVFPIYCLC SEQ ID NO: 577 RPL7L1 NM198486.2 EVWRHLLGRPHSSEQ ID NO: 578 SENP2 NM021627.2 GIFELFIL SEQ ID NO: 579 SLC29A2NM001532.2 SPCPSSPPSQPW SEQ ID NO: 580 SLC35B2 NM178148.2VLSDLGCAAGKSDDPQLWGHSHITG SEQ ID NO: 581 TH NM199293.2 HQALGAVPSCEGVSEQ ID NO: 582 WIPI2 NM001033519.1 RYGRCVHCREIVLQQPSGHRQP SEQ ID NO: 583XRRAI NM182969.1 DRKRGCCPTSSSLPISLRVRLS SEQ ID NO: 584 CIRHIA NM032830.2MTSLLSSHHPLKRRNLEP SEQ ID NO: 585 TATDN2 NM014760.3GDQQPDRTQAGLKSVSQVEDVFRELIGTQKTRTGCFPPS GS SEQ ID NO: 586 GTSEINM016426.6 VQMKMMKSSSDPLDIKKDVLLPAWN SEQ ID NO: 587 C190rf2 NM1344471GFAASWLFKKPRPSECHTVIFKEESYMN

CONCLUDING MATTER

For clarity and to ensure completeness, certain of the aspects and/orembodiments disclosed herein may be overlapping in scope, describedrepetitively, or represent recitals of the same or equivalent elementsor combinations expressed in alternative language. It will be apparentthat the choice of particular phraseology and/or of particular aspectsor elements to assert as claims involves many complex technical andlegal considerations, and no inference should be drawn that alternativedescriptions of a particular element or combination in this writtendescription necessarily do or do not encompass different subject matter;except where context otherwise requires, each described aspect orelement should be interpreted according to its own description.

It is intended that this specification be interpreted in accordance withthe normal principles of English grammar and that words and phrases begiven their ordinary English meaning as understood by persons of skillin the pertinent arts except as otherwise explicitly stated. If a word,term, or phrase is intended to be further characterized, specified, ornarrowed in some way, then additional adjectives, modifiers, ordescriptive text have been included in accordance with the normalprinciples of English grammar. It is intended that the meanings ofwords, terms, or phrases should not be modified or characterized in amanner differing from their ordinary English meaning as understood bypersons of skill in the relevant arts except on the basis of adjectives,modifiers, or descriptive text that is explicitly present.

Except as otherwise explicitly stated, terms used in this specification,including terms used in the claims and drawings, are intended as “open”terms. That is, for example, the words “including” and “comprising”should be interpreted to mean “including but not limited to,” the word“having” should be interpreted to mean “having at least,” the word“includes” should be interpreted to mean “includes but is not limitedto,” the phrases “for example” or “including by way of example” shouldbe interpreted as signifying that the example(s) given arenon-exhaustive and other examples could be given, and other similarwords and phrases should be given similar non-exclusive meanings. Exceptas explicitly stated, ordinals used as adjectives (e.g. “first object”,“second object”, etc.) in this specification, including claims anddrawing figures, are intended merely to differentiate and do not implythat any particular ordering is required. Thus, for example, unlessotherwise explicitly stated, “first measurement” and “secondmeasurement” do not imply that the first measurement necessarily takesplace before the second measurement, but merely that they are distinctmeasurements.

In the written description and appended claims, the indefinite articles“a” and/or “an” are intended to mean “at least one” or “one or more”except where expressly stated otherwise or where the enabling disclosurerequires otherwise. The word “or” as used herein is intended to mean“and/or”, except where it is expressly accompanied by the word “either”,as in “either A or B”. Applicants are aware of the provisions of 35U.S.C. §112, ¶6. The use of the words “function,” “means” or “step” inthe written description, drawings, or claims herein is not intended toinvoke the provisions of 35 U.S.C. §112, ¶6, to define the invention. Tothe contrary, if the provisions of 35 U.S.C. §112, ¶6 are sought to beinvoked, the claims will expressly include one of the exact phrases“means for performing the function of” or “step for performing thefunction of”. Moreover, even if the provisions of 35 U.S.C. §112, ¶6 areexplicitly invoked to define a claimed invention, it is intended thatthe claims not be limited only to the specific structure, material oracts that are described in the preferred embodiments, but in addition,extend to any and all structures, materials or acts that perform theclaimed function as described in alternative embodiments or forms of theinvention, or that are well known present or later-developed equivalentstructures, material or acts for performing the claimed function.

Any of the methods of the present disclosure may be implemented in wholeor part in hardware, software, or both, or by a computer program, andmay be carried out using any of the disclosed devices or apparatusaccording to any aspect or embodiment of the present invention, or inany other operable manner.

In the foregoing description, various details, specific aspects,embodiments, and examples have been described in order to illustrate andexplain the subject matter, to provide a thorough understanding of thevarious aspects, to enable persons skilled in the pertinent arts topractice the described subject matter, and to disclose the best mode ofdoing so known to applicants. These details, specific aspects,embodiments, and examples are not intended to be limiting; rather, itwill be apparent to persons of skill in the relevant arts that, basedupon the teachings herein, various changes, substitutions,modifications, rearrangements, may be made and various aspects,components, or steps may be omitted or added, without departing from thesubject matter described herein and its broader aspects. Except asotherwise expressly stated or where aspects or features are inherentlymutually exclusive, aspects and features of any embodiment describedherein may be combined with aspects and features of any one or moreother embodiments. Descriptions of theoretical principles and/orproperties, where given, are intended as explanatory and not limiting.Titles, headings, and subheadings herein and the accompanying abstractare intended merely as a convenience for locating content, and do notlimit or otherwise affect the interpretation of the content of thedisclosure. The appended claims are intended to encompass within theirscope any and all changes, substitutions, modifications, rearrangements,combinations of aspects or features, additions, and omissions that arewithin the spirit and scope of the subject matter as described hereinand/or within the knowledge of a person of skill in the art. The scopeof the invention is defined by the claims, and is not limited by or tothe particular embodiments or aspects chosen for detailed exposition inthe foregoing description, but rather extends to all embodiments oraspects as defined by the claims, as well as any equivalents of suchembodiments or aspects, whether currently known or developed in thefuture.

So as to reduce the complexity and length of the detailed description,and to provide background in certain areas of technology, each of thematerials identified in the “REFERENCES” section below is expresslyincorporated by reference. Applicants believe that the subject matterincorporated is “non-essential” in accordance with 37 CFR 1.57, becauseit is referred to for purposes of indicating the background of theinvention or illustrating the state of the art. However, if the Examinerconcludes that any of the incorporated material constitutes “essentialmaterial” within the meaning of 37 CFR 1.57(c)(1)-(3), applicants willamend the specification to expressly recite the essential material thatis incorporated by reference as allowed by the applicable rules.

REFERENCES

-   Berzofsky, J., Terabe, M., Oh, S., Belyakov, L, Ahlers, J.,    Janik, J. & Morris, J. (2004) Progress on new vaccine strategies for    the immunotherapy and prevention of cancer. J. Clin. Invest. 2004    June; 113(11): 1515-1525.-   Gite, S., Lim, M., Carlson, R., Olejnik, J., Zehnbauer, B. &    Rothschild, K. (2003) A high-throughput nonisotopic protein    truncation test. Nature Biotechnology: 21(2): 194-197.-   Kerr, C. (2002) Huntington's disease provides cancer clues. The    Lancet. Oncology: 3(9): 518.-   Leaf, C. (2004) Why we're losing the war on cancer. Fortune: 149(6):    76-82:-   Lewis, J. (2004) Therapeutic cancer vaccines: using unique antigens.    PNAS: 101 Suppl 2:14653-6.-   Linnebacher, M., Gebert, J., Rudy, W., Woerner, S., Yuan, Y.,    Bork, P. & von Knebel Doeberitz, M. (2001) Frameshift    peptide-derived T-cell epitopes: a source of novel tumor-specific    antigens. Int. J. Cancer. 93(1): 6-11.-   Saeterdal, I., Bjorheim, J., Lislerud, K., Gjertsen, M., Bukholm, L,    Olsen, O., Nesland, J., Eriksen, J., Moller, M., Lindblom, A., &    Gaudernack, G. (2001) Frameshift-mutation-derived peptides as    tumor-specific antigens in inherited and spontaneous colorectal    cancer. PNAS: 98(23):13255-60.-   Sorensen, S. A., Fenger, K. & Olsen, J. (1999) Significantly lower    incidence of cancer among patients with Huntington disease. Cancer:    86(7):1342-6.-   Sykes, K. F., and S. A. Johnston. (1999) Linear expression elements:    a rapid, in vivo, method to screen for gene functions. Nat    Biotechnol 17(4):355-9.-   Wang, R., Parkhurst, M., Kawakami, Y., Robbins, P. &    Rosenberg, S. A. (1996) Utilization of an alternative open reading    frame of a normal gene in generating a novel human cancer antigen.    The Journal of Experimental Medicine: 183, 1131-1140.

I claim:
 1. A synthetic or recombinant peptide component for ananti-cancer vaccine for administration to a member of a population of amammalian species, comprising a continuous amino acid chain, or acontinuous portion thereof from 8 to 40 amino acids in length, accordingto the formula D1-D2, wherein D1 and D2 each have at least 90% sequenceidentity to a polypeptide sequence encoded by a different exon orcontinuous portion thereof of the genome of the mammalian species,wherein D1 is encoded in a wild type reading frame and D2 is encoded ina non-wild type reading frame.
 2. The peptide component of claim 1,wherein the portion of D1 immediately adjacent to D2 is not amicrosatellite or portion thereof.
 3. The peptide component of claim 1,wherein neither of the different exons is an oncogene.
 4. The peptidecomponent of claim 1, wherein the RefSeq of the mammalian species doesnot contain a continuous nucleic acid sequence encoding the sequence ofthe peptide component.
 5. The peptide component of claim 1, wherein thenormal transcriptome of the mammalian species does not contain acontinuous nucleic acid sequence encoding the sequence of the peptidecomponent.
 6. The peptide component of claim 1, wherein the peptidecomponent aligns with at least 90% identity to all or a portion of anmRNA transcript expressed in at least one cancer type of the mammalianspecies.
 7. The peptide component of claim 1, wherein the peptidecomponent has a cancer association ratio of at least 2:1 with respect toat least one cancer type of the mammalian species.
 8. The peptidecomponent of claim 1, wherein the peptide component has a cancer serumrecognition percentage of at least 40% with respect to at least onecancer type of the mammalian species.
 9. The peptide component of claim1, wherein the peptide component is capable of being displayed in aClass I major histocompatibiity complex (MHC-I) of a type expressed byat least 4 percent of the population.
 10. The peptide component of claim1, wherein the mammalian species is mouse and the continuous amino acidchain has at least 90% sequence identity to a sequence selected from:peptide 1-78 (SEQ ID NO: 294), peptide 6-21 (SEQ ID NO:291), peptideSMC1A (SEQ ID NO: 297), peptide RBM (SEQ ID NO: 232), or peptide THAP2(SEQ ID NO: 238).
 11. The peptide component of claim 1, wherein themammalian species is human and the continuous amino acid chain has atleast 90% sequence identity to a sequence selected from: peptide 1-78(SEQ ID NO: 293), peptide 6-21 (SEQ ID NO: 290), peptide SMC1A (SEQ IDNO: 296), peptide RBM (SEQ ID NO: 234), or peptide THAP2 (SEQ ID NO:240).
 12. The peptide component of claim 1, wherein the mammalianspecies is dog and the continuous amino acid chain has at least 90%sequence identity to a sequence selected from: peptide 1-78 (SEQ ID NO:295), peptide 6-21 (SEQ ID NO: 292), peptide SMC1A (SEQ ID NO: 298),peptide RBM (SEQ ID NO: 236), or peptide THAP2 (SEQ ID NO: 242).
 13. Ananti-cancer vaccine for administration to a member of a population of amammalian species, comprising a plurality of peptide componentsaccording to claim 1, and a pharmaceutically acceptable carrier.
 14. Amethod comprising administering to a mammal an effective dose of theanti-cancer vaccine of claim
 13. 15. An anti-cancer vaccine foradministration to a member of a population of a mammalian species,comprising a synthetic or recombinant nucleic acid or plurality thereofencoding a plurality of peptide components according to claim 1, and acarrier adapted for administration of the anti-cancer vaccine by geneticimmunization.
 16. An anti-cancer vaccine according to claim 15, whereinthe plurality of peptide components comprises a first peptide componentdisplayable in a first MHC type expressed in a first cohort of thepopulation and a second peptide component displayable in a second MHCtype expressed in a second cohort of the population, and the first andsecond cohort together are more numerous by at least 2 percent of thepopulation than either cohort taken separately.
 17. A method comprisingadministering to a mammal by genetic immunization an effective dose ofthe anti-cancer vaccine of claim
 15. 18. A method comprisingadministering to a mammal by genetic immunization an effective dose ofthe anti-cancer vaccine of claim
 16. 19. A method comprising: (a)administering to a mammal by genetic immunization an effective dose ofan anti-cancer vaccine comprising a synthetic or recombinant nucleicacid or plurality thereof, encoding a plurality of peptide componentsaccording to claim 1, and a carrier adapted for administration of theanti-cancer vaccine by genetic immunization; and (b) administering tothe mammal an effective dose of an anti-cancer vaccine comprising atleast one peptide component according to claim 1 having a sequence atleast 90% identical to at least one of the plurality of peptidecomponents encoded by the nucleic acid or plurality thereof of step (a),in a pharmaceutically acceptable carrier.
 20. The method of claim 19,wherein step (b) is performed at least two weeks after step (a).